CN117561078A

CN117561078A - Biomarkers for assessing risk of developing acute covd-19 and post-acute covd-19

Info

Publication number: CN117561078A
Application number: CN202280026844.6A
Authority: CN
Inventors: G·A·德莫普洛斯; T·杜德勒; N·J·林奇; H-W·施瓦布尔; K·谢弗; 矢吹宗久
Original assignee: Omeros Medical Systems Inc
Current assignee: Omeros Corp
Priority date: 2021-02-05
Filing date: 2022-02-04
Publication date: 2024-02-13

Abstract

Disclosed herein are compositions, kits, and methods for determining the concentration of liquid phase MASP-2/C1-INH complex in a biological fluid, e.g., obtained from a subject infected with SARS-CoV-2. Also disclosed are methods of using the compositions, methods and kits for detecting MASP-2/C1-INH complexes to determine the status of lectin pathway activation in a mammalian subject and thereby assessing the risk of developing a COVID-19-associated ARDS or other adverse outcome in a subject infected or already infected with SARS-CoV-2, or to determine the need for treatment or efficacy of treatment with a complement inhibitor, e.g., a MASP-2 inhibitor, in a subject in need thereof.

Description

Biomarkers for assessing risk of developing acute covd-19 and post-acute covd-19

Statement regarding sequence listing

The sequence listing associated with this application is provided in text format in place of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named mp_1_0319_pct_sequence_listing_20220131_st25.Txt. The text file is 147KB; created at 2022, 2, 1; and submitted via FS-Web together with the submission of the specification.

Background

The complement system provides an early mechanism of action in humans and other vertebrates to initiate, amplify, and coordinate immune responses to microbial infections and other acute injuries (m.k.liszewski and j.p. atkinson,1993, in Fundamental Immunology, third edition, edited by w.e.Paul, raven Press, ltd., new York). Although complement activation provides a valuable first line defense against potential pathogens, complement activity that promotes a protective immune response may also represent a potential threat to the host (K.R. Kalli et al, springer Semin. Immunopathol.15:417-431, 1994;B.P.Morgan,Eur.J.Clinical Investig.24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Although activated neutrophils are essential for host defense, they are indistinguishable in terms of their release of destructive enzymes and may cause organ damage. In addition, complement activation may cause deposition of lytic complement components on nearby host cells as well as microbial targets, resulting in host cell lysis.

Currently, it is widely accepted that the complement system can be activated by three different pathways: classical pathway, lectin pathway and alternative pathway. The classical pathway is usually triggered by complexes consisting of host antibodies that bind to foreign particles (i.e., antigens), thus requiring prior exposure to the antigen to generate a specific antibody response. Since activation of the classical pathway depends on the host's previous adaptive immune response, the classical pathway is part of the adaptive immune system. In contrast, both lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

Lectin pathway is widely believed to have a major role in host defense against infection in the host first used in the experiment. Strong evidence that MBL is involved in host defense comes from analysis of patients with reduced serum levels of functional MBL (Kilpatrick, biochem. Biophys. Acta1572:401-413, (2002)). Such patients exhibit sensitivity to recurrent bacterial and fungal infections. These symptoms are usually apparent early in life, during the apparent vulnerability window due to reduced maternal derived antibody titers, but before the complete repertoire of antibody responses (repotoire) develops. This syndrome often results from mutations at several sites in the collagenous portion of MBL that interfere with the correct formation of MBL oligomers. However, since MBL can act as an opsonin independent of complement, it is not known to what extent the increase in infection susceptibility is due to impaired complement activation.

All three pathways (i.e., classical pathway, lectin pathway, and alternative pathway) have been thought to converge at C5, which C5 is cleaved to form products with multiple pro-inflammatory effects. The convergent pathway has been termed the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing smooth muscle and vascular tone and vascular permeability changes. It is also a powerful chemokine and activator of both neutrophils and monocytes. C5 a-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. C5 cleavage results in the formation of C5b-9, also known as a Membrane Attack Complex (MAC). There is strong evidence that, in addition to the role of forming complexes as cleavage pores, sub-cleavage MAC deposition may play an important role in inflammation.

In addition to the necessary role in immune defenses, the complement system contributes to tissue damage in many clinical situations. Although there is extensive evidence suggesting that both the classical and alternative complement pathways are involved in the pathogenesis of non-infectious human diseases, the role of the lectin pathway has just begun to be assessed. Recent studies provide the following evidence: activation of the lectin pathway may be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Cold et al (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and showed deposition of C3 after exposure to human serum (Cold et al, am. J. Pathol.156:1549-1556, (2000)). In addition, treatment of human serum with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complement activation. These findings extend to a rat model of myocardial ischemia reperfusion in which rats treated with blocking antibodies to rat MBL showed significantly less myocardial damage following coronary occlusion compared to rats treated with control antibodies (Jordan et al Circulation104:1413-1418, (2001)). The molecular mechanism by which MBL binds to vascular endothelium after oxidative stress is not yet known; recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by binding of MBL to vascular endothelial cytokeratin (rather than glycoconjugates) (Cold et al, am. J. Pathol.159:1045-1054, (2001)). Other studies have suggested that the classical and alternative pathways are involved in the pathogenesis of ischemia/reperfusion injury, while the role of the lectin pathway in this disease remains controversial (Riedermann, n.c. et al, am.j. Pathol.162:363-367, 2003).

Fibrosis is the excessive formation of connective tissue in organs or tissues, usually in response to injury or injury. The hallmark of fibrosis is the overproduction of extracellular matrix following local trauma. Normal physiological responses to injury lead to deposition of connective tissue, but this initially beneficial repair process may persist and become pathologic, altering the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia. The influx of inflammatory cells, including macrophages and lymphocytes, results in cytokine release and the deposition of collagen, fibronectin and other molecular markers of fibrosis is amplified. Conventional therapeutic approaches have largely targeted the inflammatory process of fibrosis using corticosteroids and immunosuppressive drugs. Unfortunately, these anti-inflammatory agents have little to no clinical effect. There is no effective treatment or therapeutic for fibrosis at present, but both animal studies and biographical reports suggest that fibrotic tissue damage can be reversed (Tampe and Zeisberg, nat Rev Nephrol, vol. 10: 226-237, 2014).

The kidneys have limited ability to recover from injury. Various renal pathological conditions lead to localized inflammation, which causes scarring and fibrosis of the kidney tissue. The persistence of inflammatory stimuli drives the inflammation and fibrosis of the tubular interstitium and progressive renal function impairment in chronic kidney disease. Its progression to end-stage renal failure is associated with significant morbidity and mortality. Since tubular interstitial fibrosis is a common endpoint of a variety of renal pathological conditions, it represents a key target for therapies aimed at preventing renal failure. Risk factors (e.g., proteinuria) independent of primary kidney disease contribute to the development of renal fibrosis and loss of renal excretion function by driving local inflammation, thereby enhancing disease progression.

Given the role of fibrosis in many diseases and disorders, such as tubular interstitial fibrosis leading to chronic kidney disease, there is an urgent need to develop therapeutically effective agents for treating diseases and conditions caused or exacerbated by fibrosis. Further in view of the lack of new and existing therapies targeting inflammatory pro-fibrotic pathways in kidney disease, there is a need to develop therapeutically effective agents to treat, inhibit, prevent and/or reverse kidney fibrosis, and thereby prevent progressive chronic kidney disease.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2 or SARS-CoV-2), a virus closely related to SARS virus (world health organization (World Health Organization), 2/11/2020,Novel Coronavirus Situation Report 22). Those affected by covd-19 may develop fever, dry cough, fatigue, and shortness of breath. Cases may progress to respiratory dysfunction, including pneumonia, severe acute respiratory syndrome, and death in the weakest person (see, e.g., hui d.s. Et al, int J effect Dis 91:264-266, 14 days 1 month 2020). There is no vaccine or specific antiviral therapy, which manages treatments involving symptoms and supportive care.

Influenza (also known as "influenza") is an infectious disease caused by RNA influenza viruses. Symptoms of influenza virus infection can be mild to severe and include high fever, runny nose, sore throat, muscle and joint pain, headache, cough, and tiredness. These symptoms typically begin two days after exposure to the virus and most last for less than a week, however, cough may last for more than two weeks. (see "Influenza Seasonal, world Health Organization, 2018, 11, 6). Complications of influenza may include viral pneumonia, acute Respiratory Distress Syndrome (ARDS), secondary bacterial pneumonia, sinus infections, and exacerbation of previous health problems such as asthma or heart failure (see "Key Facts About Influenza (Flu)" Centers for Disease Control and Prevention (CDC), 9, 2014). The effects of influenza are much more severe than the common cold and last longer. Most people recover completely in about one to two weeks, but others develop life-threatening complications such as pneumonia. Thus, influenza can be fatal, especially in infirm, young and old with an impaired immune system or long-term illness. See Hilleman MR, vaccine.20 (25-26): 3068-87 (2002).

Three of the four types of influenza viruses affect humans: type a, type b and type c (see "Types of Influenza Viruses Seasonal Influenza (Flu), centers for Disease Control and Prevention (CDC). 2017, 9, 27). Butyl is not known to infect humans, but is believed to have such potential (see "Novel Influenza D virus: epidemic, virology, evolution and biological characteristics," virolence.8 (8): 1580-91, 2017). The serotypes of influenza a that have been identified in humans are: H1N1 (causing "spanish influenza" in 1918 and "swine influenza" in 2009); H2N2 (causing "asian influenza" in 1957), H3N2 (causing "hong kong influenza" in 1968), H5N1 (causing "avian influenza" in 2004), H7N7, H1N2, H9N2, H7N3, H10N7, H7N9, and H6N1. See world health organization (6, 30, 2006). "Epidemiology of WHO-confirmed human cases of avian influenza A (H5N 1) input, wkly epidemic Rec.81 (26): 249-57; fouchier RA et al (2004) PNAS 101 (5): 1356-61; wkly epidemic Rec.83 (46): 415-20,Asian Lineage Avian Influenza A (H7N 9) Virus, centers for Disease Control and Prevention (CDC), 12 months and 7 days 2018.

Common symptoms of influenza viruses (also known as influenza), such as fever, headache, and fatigue, are the result of a large number of pro-inflammatory cytokines and chemokines (e.g., interferons or tumor necrosis factors) produced by influenza-infected cells. See Eccles R. Et al Lancet Infect Dis5 (11): 718-25 (2005); schmitz N et al Journal of virology 79 (10): 6441-8 (2005). This large-scale immune response may lead to life-threatening cytokine storms. This effect has been proposed as a cause of abnormal mortality in both H5N1 avian influenza and 1918 pandemic strains. Cheung CY et al, lancet.360 (9348): 1831-37 (2002); kash JC et al, nature.443 (7111): 578-81 (2006). Influenza also appears to trigger programmed cell death (apoptosis), see Clinical Respiratory Medicine, elsevier Health sciences, page 311 (2012).

Accordingly, there is an urgent need to develop therapeutically effective agents to treat, inhibit and/or prevent coronavirus-induced pneumonia and acute respiratory distress syndrome, as well as influenza virus-induced pneumonia and acute respiratory distress syndrome.

Furthermore, in order to maximize successful treatment and protection of people from covd-19, biomarkers and highly accurate tests are urgently needed to identify those at risk of developing acute covd-19 and/or long-term disease (post-course covd-19), otherwise known as long-covd-19 syndrome, or who have developed a protective immune response against the covd-19 disease response. There is also a need for testing to determine the efficacy of therapeutic agents to treat and/or prevent complications associated with covd-19 in subjects infected with SARS-CoV-2, including those suffering from or at risk of developing long-covd-19.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the invention provides a method for treating, inhibiting, reducing or preventing acute respiratory distress syndrome, pneumonia or some other acute manifestations of covd-19, such as thrombosis, in a mammalian subject infected with SARS-CoV-2 comprising (i) determining the level of MASP-2/C1-INH complex in a biological sample obtained from said subject, wherein an increased level of MASP-2/C1-INH complex, as compared to a healthy control sample or other reference standard, indicates an increased risk of developing one or more acute manifestations of covd-19; and (ii) administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation to the subject having an increased level of the MASP-2/C1-INH complex. In some embodiments, the subject has one or more respiratory symptoms, and the method comprises administering to the subject an amount of a MASP-2 inhibitor effective to ameliorate at least one respiratory symptom (i.e., improve respiratory function). In one embodiment, the MASP-2 inhibitor is a MASP-2 antibody or antigen binding fragment thereof. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO. 6. In one embodiment, the MASP-2 inhibitor selectively inhibits aggregation The hormone pathway complement activation without substantially inhibiting C1 q-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a small molecule that inhibits MASP-2 dependent complement activation, such as a synthetic or semi-synthetic small molecule. In one embodiment, the MASP-2 inhibitor is an inhibitor of MASP-2 expression. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum, wherein IC ₅₀ 30nM or less. In one embodiment, the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence as set forth in SEQ ID NO. 67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence as set forth in SEQ ID NO. 69. In one embodiment, the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69.

In another aspect, the invention provides a method for treating, reducing, preventing or reducing the risk of developing one or more covd-19 associated long-term sequelae in a mammalian subject that has been infected with SARS-CoV-2, comprising (i) determining the level of MASP-2/C1-INH complex in a biological sample obtained from the subject, wherein an increased level of MASP-2/C1-INH complex, as compared to a healthy control sample, indicates an increased risk of developing one or more covd-19 associated long-term sequelae; and (ii) administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation to the subject having an increased level of the MASP-2/C1-INH complex. In one embodiment, the MASP-2 inhibitor is a MASP-2 antibody or antigen thereofBinding fragments. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO. 6. In one embodiment, the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1 q-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a small molecule that inhibits MASP-2 dependent complement activation, such as a synthetic or semi-synthetic small molecule. In one embodiment, the MASP-2 inhibitor is an inhibitor of MASP-2 expression. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum, wherein IC ₅₀ 30nM or less. In one embodiment, the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence as set forth in SEQ ID NO. 67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence as set forth in SEQ ID NO. 69. In one embodiment, the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69.

In another aspect, the present disclosure provides a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human MASP-2 complexed with C1-INH, wherein the antibody comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO. 87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO. 88, wherein the CDRs are numbered according to the Kabat numbering system. In one embodiment, the MASP-2 specific antibody comprises a heavy chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 87 and a light chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 88. In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof is labeled with a detectable moiety, such as a detectable moiety suitable for use in an immunoassay, as further described herein. In one embodiment, the MASP-2 specific antibody or fragment thereof is immobilized on a substrate, e.g., a substrate suitable for use in an immunoassay, e.g., an immunoassay for detecting MASP-2/C1-INH complex.

In another aspect, the present disclosure provides a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human MASP-2 complexed with C1-INH, wherein the antibody comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO. 97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO. 98, wherein the CDRs are numbered according to the Kabat numbering system. In one embodiment, the MASP-2 specific antibody comprises a heavy chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 97 and a light chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 98. In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof is labeled with a detectable moiety, such as a detectable moiety suitable for use in an immunoassay, as further described herein. In one embodiment, the MASP-2 specific antibody or fragment thereof is immobilized on a substrate, e.g., a substrate suitable for use in an immunoassay, e.g., an immunoassay for detecting MASP-2/C1-INH complex.

In another aspect, the present disclosure provides a method of measuring the amount of MASP-2/C1-INH in a biological sample, comprising: (a) providing a test biological sample from a human subject; (b) Performing an immunoassay comprising capturing and detecting MASP-2/C1-INH complexes in a test sample, wherein MASP-2/C1-INH complexes are captured with monoclonal antibodies that specifically bind to human MASP-2; and detecting MASP-2/C1-INH complex directly or indirectly with an antibody that specifically binds to C1-INH; and (C) comparing the level of MASP-2/C1-INH complex detected according to (b) with a predetermined level or a control sample, wherein the level of MASP-2/C1-INH complex detected in the test sample is indicative of the extent of lectin pathway complement activation. In some embodiments, the biological sample is a fluid sample from a human subject selected from the group consisting of whole blood, serum, plasma, urine, and cerebrospinal fluid. In some embodiments, the human subject is currently infected with SARS-CoV-2, or has been previously infected with SARS-CoV-2. In some embodiments, an antibody that specifically binds MASP-2 comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as shown in SEQ ID NO:88, wherein the CDRs are numbered according to the Kabat numbering system. In some embodiments, an antibody that specifically binds MASP-2 comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

In another aspect, the present disclosure provides a method of determining the risk of a subject infected or having been infected with SARS-CoV-2 to develop a COVID-19-associated ARDS or a COVID-19-associated long-term sequelae, comprising: (a) obtaining a biological sample from the subject; (b) Measuring the level of MASP-2/C1-INH complex in the sample; (c) Comparing the measured level with a predetermined level of MASP-2/C1-INH complex or a reference standard to assess the risk of developing ARDS associated with COVID-19 and/or long-term sequelae associated with COVID-19; and (d) determining the risk of the subject developing a covd-19 related ARDS and/or a long-term sequelae associated with covd-19 and reporting the result to a patient, physician or database; (e) Optionally, the treatment is administered to a subject determined to be likely to develop an acute disease and/or long-term sequelae associated with a covd-19 infection. In some embodiments, the level of MASP-2/C1-INH complex is measured in an immunoassay. In some embodiments, step (b) comprises performing an immunoassay, e.g., an ELISA assay, to measure the level of MASP-2/C1-INH complex in the biological sample. In some embodiments, the immunoassays include the use of antibodies that specifically bind MASP-2, the antibodies comprising binding domains that comprise HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO. 87 and that comprise LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as shown in SEQ ID NO. 88, wherein the CDRs are numbered according to the Kabat numbering system. In some embodiments, the immunoassays include the use of antibodies that specifically bind MASP-2, the antibodies comprising binding domains that comprise HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO. 97 and that comprise LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as shown in SEQ ID NO. 98, wherein the CDRs are numbered according to the Kabat numbering system.

In another aspect, the present disclosure provides a method for monitoring the efficacy of treatment with a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, in a mammalian subject in need thereof, the method comprising: (a) Administering a dose of a MASP-2 inhibitory antibody or antigen-binding fragment thereof to a mammalian subject at a first time point; (b) Assessing a first level of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (a); (c) Treating the subject with the MASP-2 inhibitory antibody or antigen-binding fragment thereof at a second time point; (d) Assessing a second level of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (C); and (e) comparing the level of MASP-2/C1-INH complex assessed in step (b) with the level of MASP-2/C1-INH complex assessed in step (d) to determine the efficacy of the MASP-2 inhibitory antibody or antigen binding fragment thereof in the mammalian subject. In some embodiments, the subject is a human subject having or at risk of developing COVID-19 or a long-term sequelae associated with COVID-19. In some embodiments, the subject is a human subject having or at risk of developing a disease or disorder selected from HSCT-TMA, igAN, lupus nephritis, and graft versus host disease or some other lectin pathway disease or disorder. In some embodiments, the level of MASP-2/C1-INH complex is measured in an immunoassay. In some embodiments, step (b) comprises performing an immunoassay, e.g., an ELISA assay, to measure the level of MASP-2/C1-INH complex in the biological sample. In some embodiments, the immunoassays include the use of antibodies that specifically bind MASP-2, the antibodies comprising binding domains that comprise HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO. 87 and that comprise LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as shown in SEQ ID NO. 88, wherein the CDRs are numbered according to the Kabat numbering system. In some embodiments, the immunoassays include the use of antibodies that specifically bind MASP-2, the antibodies comprising binding domains that comprise HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO. 97 and that comprise LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as shown in SEQ ID NO. 98, wherein the CDRs are numbered according to the Kabat numbering system.

Drawings

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a diagram showing the genomic structure of human MASP-2;

FIG. 2A is a schematic diagram showing the domain structure of human MASP-2 protein;

FIG. 2B is a schematic diagram showing the domain structure of human MAp19 protein;

FIG. 3 is a diagram showing a murine MASP-2 knockout strategy;

FIG. 4 is a diagram showing a human MASP-2 minigene (minigene) construct;

FIG. 5A presents results demonstrating that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation as measured by lack of C4b deposition on mannans as described in example 2;

FIG. 5B presents results demonstrating that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation as measured by lack of C4B deposition on zymosan as described in example 2;

FIG. 5C presents results demonstrating the relative C4 activation levels of serum samples obtained from MASP-2+/-, MASP-2-/-and wild-type strains as measured by C4b deposition on mannans and zymosan as described in example 2;

FIG. 6 presents results demonstrating that addition of murine recombinant MASP-2 to a MASP-2-/-serum sample restores lectin pathway-mediated C4 activation in a protein concentration-dependent manner as measured by C4b deposition on mannans as described in example 2;

FIG. 7 presents results demonstrating that the classical pathway is functional in MASP-2-/-strains as described in example 8;

FIG. 8A presents results demonstrating that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation as described in example 10;

FIG. 8B presents results demonstrating that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2 as described in example 10;

FIG. 8C presents results demonstrating that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage as described in example 10;

the results presented in FIG. 9 demonstrate that all anti-MASP-2 Fab2 antibodies tested that inhibit the formation of C3 convertase were also found to inhibit C4 cleavage as described in example 10;

FIG. 10 is a diagram showing recombinant polypeptides derived from rat MASP-2 for epitope mapping of MASP-2 blocking Fab2 antibodies, as described in example 11;

FIG. 11 presents results demonstrating the binding of anti-MASP-2 Fab2#40 and #60 to rat MASP-2 polypeptides as described in example 11;

FIG. 12A graphically illustrates the level of MAC deposition in the presence or absence of human MASP-2 monoclonal antibody (OMS 646) under lectin pathway specific assay conditions, confirming that OMS646 inhibits lectin-mediated MAC deposition, IC thereof ₅₀ Values of about 1nM, as described in example 12;

FIG. 12B graphically illustrates the level of MAC deposition in the presence or absence of human MASP-2 monoclonal antibody (OMS 646) under classical pathway-specific assay conditions, confirming that OMS646 does not inhibit classical pathway-mediated MAC deposition, as described in example 12;

FIG. 12C graphically illustrates the level of MAC deposition in the presence or absence of human MASP-2 monoclonal antibody (OMS 646) under alternative pathway-specific assay conditions, confirming that OMS646 does not inhibit alternative pathway-mediated MAC deposition, as described in example 12;

figure 13 graphically illustrates the Pharmacokinetic (PK) profile of human MASP-2 monoclonal antibody (OMS 646) in mice showing OMS646 concentration (n=3 animals/group average) as a function of time after administration at indicated doses, as described in example 12;

FIG. 14A graphically illustrates the Pharmacodynamic (PD) response of human MASP-2 monoclonal antibody (OMS 646) measured in mice as a decrease in systemic lectin pathway activity following intravenous administration, as described in example 12;

FIG. 14B graphically illustrates the Pharmacodynamic (PD) response of human MASP-2 monoclonal antibody (OMS 646) measured in mice as a decrease in systemic lectin pathway activity following subcutaneous administration, as described in example 12;

FIG. 15 graphically illustrates results of computer-based image analysis of kidney tissue sections stained with sirius red, wherein the tissue sections were wild-type and MASP-2-/-mice after 7 days of Unilateral Ureteral Obstruction (UUO), and sham-operated wild-type and MASP-2-/-mice, as described in example 14;

FIG. 16 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with F4/80 macrophage specific antibody, wherein the tissue sections were wild-type and MASP-2-/-mice, as well as sham-operated wild-type and MASP-2-/-mice, 7 days after unilateral ureteral obstruction (UUFO), as described in example 14.

Figure 17 graphically illustrates the relative mRNA expression levels of collagen-4 in kidney tissue sections obtained from wild-type and MASP-2-/-mice after 7 days of Unilateral Ureteral Obstruction (UUO), as well as sham-operated wild-type and MASP-2-/-mice, as measured by quantitative PCR (qPCR), as described in example 14.

FIG. 18 graphically illustrates the relative mRNA expression levels of transforming growth factor beta-1 (TGF beta-1) in kidney tissue sections from wild-type and MASP-2-/-mice after 7 days from unilateral ureteral obstruction (UUFO), as well as sham-operated wild-type and MASP-2-/-mice, as measured by qPCR, as described in example 14.

FIG. 19 graphically illustrates relative mRNA expression levels of interleukin 6 (IL-6) in kidney tissue sections from wild-type and MASP-2-/-mice after 7 days from unilateral ureteral obstruction (UUFO), as well as sham-operated wild-type and MASP-2-/-mice, as measured by qPCR, as described in example 14.

Figure 20 graphically illustrates the relative mRNA expression levels of interferon-gamma in kidney tissue sections from wild-type and MASP-2-/-mice after 7 days from Unilateral Ureteral Obstruction (UUO), as well as sham-operated wild-type and MASP-2-/-mice, as measured by qPCR, as described in example 14.

Figure 21 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with sirius red obtained from wild-type mice treated with MASP-2 inhibitory antibodies as well as isotype control antibodies 7 days after Unilateral Ureteral Obstruction (UUO), as described in example 15.

Figure 22 graphically illustrates the hydroxyproline content of kidneys harvested 7 days after Unilateral Ureteral Obstruction (UUO) from wild-type mice treated with MASP-2 inhibitory antibodies, as compared to hydroxyproline levels in tissues from obstructed kidneys from wild-type mice treated with IgG4 isotype control, as described in example 15.

Figure 23 graphically shows the total serum protein (mg/ml) measured at day 15 of the protein overload study in wild-type control mice (n=2), BSA-receiving wild-type mice (n=6) and BSA-receiving MASP-2-/-mice (n=6) alone in saline, as described in example 16.

Figure 24 graphically illustrates total amount of excreted protein (mg) in urine collected over a 24 hour period at day 15 of the protein overload study from wild type control mice receiving only saline (n=2), wild type BSA (n=6) and MASP-2-/-mice receiving BSA (n=6), as described in example 16.

Fig. 25 shows representative hematoxylin and eosin (H & E) -stained kidney tissue sections from the following groups of mice at day 15 of the protein overload study: (Panel A) wild-type control mice; (Panel B) MASP-2-/-control mice, (Panel C) wild-type mice treated with BSA; and (panel D) MASP-2-/-mice treated with Bovine Serum Albumin (BSA) as described in example 16.

Figure 26 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with macrophage specific antibody F4/80, showing the mean staining area (%) of macrophages, wherein the tissue sections were obtained from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2-/-mice treated with BSA (n=5) on day 15 of the protein overload study, as described in example 16.

Figure 27A graphically illustrates analysis of the presence of macrophage-proteinuria correlation in each wild-type mouse treated with BSA (n=6) by plotting total excreted protein measured in urine from 24 hours samples against macrophage infiltration (average stained area%) as described in example 16.

Figure 27B graphically illustrates analysis of the presence of macrophage-proteinuria correlation in each MASP-2-/-mouse treated with BSA (n=5) by plotting total excreted protein in urine of 24 hour samples against macrophage infiltration (average stained area%) as described in example 16.

Figure 28 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tgfβ antibodies (measured as% tgfβ antibody staining area) in wild-type mice treated with BSA (n=4) and MASP-2-/-mice treated with BSA (n=5), as described in example 16.

Figure 29 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tnfα antibodies (measured as% tnfα antibody staining area) in wild type mice treated with BSA (n=4) and MASP-2-/-mice treated with BSA (n=5), as described in example 16.

Figure 30 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as% IL-6 antibody staining area) in wild type control mice, MASP-2-/-control mice, wild type mice treated with BSA (n=7), and MASP-2-/-mice treated with BSA (n=7), as described in example 16.

Figure 31 graphically illustrates the frequency of TUNEL apoptotic cells counted in 20 High Power Fields (HPF) selected consecutively from tissue sections from renal cortex in wild type control mice (n=1), MASP-2-/-control mice (n), BSA treated wild type mice (n=7) and BSA treated MASP-2-/-mice (n=7), as described in example 16.

Fig. 32 shows representative H & E stained tissue sections from the following mice groups on day 15 after treatment with BSA: (Panel A) wild-type control mice treated with saline, (Panel B) isotype antibody-treated control mice, and (Panel C) wild-type mice treated with MASP-2 inhibitory antibodies, as described in example 17.

Figure 33 graphically illustrates the frequency of TUNEL apoptotic cells counted in 20 High Power Fields (HPF) selected consecutively from tissue sections from renal cortex in wild-type mice treated with saline control and BSA (n=8), wild-type mice treated with isotype control antibodies and BSA (n=8), and wild-type mice treated with MASP-2 inhibitory antibodies and BSA (n=7), as described in example 17.

Figure 34 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tgfβ antibodies (measured as% tgfβ antibody staining area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibodies (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibodies (n=8), as described in example 17.

Figure 35 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tnfα antibodies (measured as% tnfα antibody staining area) in wild type mice treated with BSA and saline (n=8), wild type mice treated with BSA and isotype control antibodies (n=7), and wild type mice treated with BSA and MASP-2 inhibitory antibodies (n=8), as described in example 17.

Figure 36 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as% IL-6 antibody staining area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibodies (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibodies (n=8), as described in example 17.

Fig. 37 shows representative H & E stained tissue sections from the following groups of mice on day 14 after treatment with doxorubicin alone or saline (control): (panels A-1, A-2, A-3) wild-type control mice treated with saline alone; (panels B-1, B-2, B-3) wild-type mice treated with doxorubicin; and (panels C-1, C-2, C-3) MASP-2-/-mice treated with doxorubicin, as described in example 18;

figure 38 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with macrophage specific antibody F4/80, showing the average staining area (%) of macrophages from the following mice groups on day 14 after treatment with doxorubicin alone or saline (wild type control): wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2-/-mice treated with saline alone, and MASP-2/-mice treated with doxorubicin, wherein p = 0.007, as described in example 18;

Figure 39 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with sirius red, showing the areas of staining (%) of collagen deposition from the following groups of mice on day 14 after treatment with doxorubicin alone or saline (wild type control): wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2-/-mice treated with saline alone, and MASP-2/-mice treated with doxorubicin, wherein p = 0.005, as described in example 18; and

figure 40 graphically illustrates urinary albumin/creatinine ratio (uACR) in two IgA patients during the course of twelve weeks of weekly study with MASP-2 inhibitory antibodies (OMS 646), as described in example 19.

FIG. 41A shows representative images (H & E,400 x) of immunohistochemical analysis of septal vascular tissue sections from the lungs of a patient with COVID-19, as described in example 21.

FIG. 41B shows representative images (H & E,400 x) of immunohistochemical analysis of septal vascular tissue sections from the lungs of a patient with COVID-19, as described in example 21.

FIG. 41C shows representative images of immunohistochemical analysis of tissue sections of medium diameter pulmonary septal vessels from a patient with COVID-19, as described in example 21.

FIG. 41D shows representative images (H & E,400 x) of immunohistochemical analysis of liver parenchymal tissue sections from a patient with COVID-19, as described in example 21.

Figure 42A graphically illustrates the Circulating Endothelial Cells (CEC)/ml counts in peripheral blood of a normal healthy control (n-6) compared to CEC/ml counts in a covd-19 patient (n=33) that is not part of this study, as described in example 21.

Figure 42B graphically shows CEC/ml counts in 6 patients selected for this study before (baseline) and after treatment with nasoprolimab Li Shan, boxes representing values from the first quartile to the third quartile, horizontal lines showing median values, and whisker lines indicating minimum and maximum values, as described in example 21.

FIG. 43 graphically illustrates serum levels of C-reactive protein (CRP) (median; quartile range (IQR)) in 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naxol Li Shan, as described in example 21.

FIG. 44 graphically illustrates serum levels of Lactate Dehydrogenase (LDH) (median; IQR) in 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naline Li Shan, as described in example 21.

Figure 45 graphically illustrates serum levels (median; interquartile spacing (IQR)) of interleukin 6 (IL-6) in 6 covd-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with nano-wire Li Shan, as described in example 21.

FIG. 46 graphically illustrates serum levels (median; interquartile spacing (IQR)) of interleukin 8 (IL-8) in 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naxol Li Shan, as described in example 21.

Figure 47A shows a CT scan of patient #4, at day 5, after enrollment (i.e., after anti-treatment with nano-cable Li Shan), where the patient was observed to have severe interstitial pneumonia with diffuse ground glass shadows involving both peripheral and central areas, especially solid changes in the lower lobes of the left lung, and massive bilateral pulmonary embolism with filling defects in the intercolumenal and segmental arteries (not shown), as described in example 21.

Figure 47B shows CT scan of patient #4 at day 16 since enrollment (i.e., after anti-treatment with nano-wire Li Shan), with significantly reduced ground glass shadows and almost complete regression of the substantial reality, as described in example 21.

Figure 48 graphically illustrates serum levels of IL-6 (pg/mL) at baseline and at various time points after naloxone Li Shan anti-treatment (after 2 doses, after 4 doses) in patients treated with naloxone Li Shan, where the boxes represent the values from the first quartile to the third quartile, the horizontal line shows the median, and the points show the values for all patients, as described in example 21.

Figure 49 graphically shows serum levels of IL-8 (pg/mL) at baseline and at various time points after naloxone Li Shan anti-treatment (after 2 doses, after 4 doses) in patients treated with naloxone Li Shan, where the boxes represent the values from the first quartile to the third quartile, the horizontal line shows the median, and the points show the values for all patients, as described in example 21.

Figure 50 graphically illustrates the clinical results of 6 covd-19 infected patients treated with naloxone Li Shan as described in example 21.

FIG. 51A graphically illustrates serum levels (units/liter, U/L) of aspartate Aminotransferase (AST) before and after nano-wire Li Shan anti-treatment. The black lines represent the median and quartile spacing (IQR). The red line represents normal levels, while the dots show the values for all patients, as described in example 21.

Figure 51B graphically illustrates the serum levels of D-dimer values (ng/ml) in four patients whose baseline values were available prior to initiation of anti-treatment with nano-cable Li Shan. The black circles indicate when steroid therapy is initiated. The red line represents normal levels, as described in example 21.

Figure 52A graphically illustrates the serum levels (ng/ml) of D-dimer values in the seventh covd-19 infected patient treated with the naloxone Li Shan antibody (patient # 7) at baseline prior to treatment (day 0), and at various time points after treatment with the naloxone Li Shan antibody, wherein administration with the naloxone Li Shan antibody is indicated by the vertical arrow, and wherein the horizontal line represents normal levels, as described in example 22.

Figure 52B graphically illustrates serum levels of C-reactive protein (CRP) in patient #7 infected with COVID-19 at baseline prior to treatment (day 0), and at various time points after treatment with the naloxone Li Shan antibody, wherein administration with the naloxone Li Shan antibody is indicated by the vertical arrow, and wherein the horizontal line represents normal levels, as described in example 22.

Figure 52C graphically illustrates serum levels (units/liter, U/L) of aspartate Aminotransferase (AST) in patient #7 infected with covd-19 at baseline prior to treatment (day 0), and at various time points after naloxone Li Shan anti-treatment, wherein administration with naloxone Li Shan antibody is indicated by the vertical arrow, and wherein the horizontal line represents normal levels, as described in example 22.

Figure 52D graphically illustrates serum levels (units/liter, U/L) of alanine Aminotransferase (ALT) in patient #7 infected with covd-19 at baseline prior to treatment (day 0), and at various time points after naloxone Li Shan anti-treatment, wherein dosing with naloxone Li Shan antibody is indicated by the vertical arrow, and wherein the horizontal line represents normal levels, as described in example 22.

Figure 52E graphically illustrates serum levels of Lactate Dehydrogenase (LDH) in patient #7 with covd-19 at baseline prior to treatment (day 0), and at various time points after treatment with the naloxone Li Shan antibody, wherein administration with the naloxone Li Shan antibody is indicated by the vertical arrow, and wherein the horizontal line represents normal levels, as described in example 22.

Figure 53 graphically illustrates anti-SARS-CoV-2 antibody titers in patient #7 over time, indicating that treatment with naloxone Li Shan antibody does not block effector functions of the adaptive immune response, as described in example 22.

FIG. 54 graphically illustrates the concentration-dependent binding of recombinant MASP-2 to SARS-Cov-2 nucleocapsid protein (NP 2) as compared to the BSA control, as described in example 23.

FIG. 55 depicts an SDS-PAGE Western blot gel showing that MASP-2 binds directly to NP and cleaves C4, and that addition of MASP-2 inhibitory antibody HG4 inhibits NP/MASP-2-mediated cleavage of C4, as described in example 23.

Figure 56 graphically illustrates CH50 values in different subject populations in a longitudinal study, wherein each "x" symbol in the graph represents an individual subject, as described in example 24.

Figure 57 graphically illustrates the C5a levels (ng/ml) in plasma samples obtained from different subject populations in a longitudinal study, wherein each "x" symbol in the figure represents an individual subject, as described in example 24.

Figure 58 graphically illustrates the Bb levels (μg/mL) in plasma obtained from different subject populations in a longitudinal study, wherein each "x" symbol in the figure represents an individual subject, as described in example 24.

FIG. 59 graphically illustrates an OD-based ₄₅₀ Values, the amount of MASP-2/C1-INH complex detected with each of the four candidate anti-MASP-2 mAbs (clones C1, C7, D8 and H1) at various concentrations of activated serum is as described in example 25.

Figure 60 graphically illustrates the results of ELISA assays measuring MASP-2/C1-INH complexes in 5% serum from acute covd patients (16 samples from 3 patients <14 days after hospitalization), convalescent patients (n=15), seropositive persons (n=15), and seronegative persons (n=34), as described in example 25.

FIG. 61 graphically depicts the amount of MASP-2/C1-INH complex present in 3 acute COVID-19 patients (# 2, #3, and # 4) after admission and for a period of up to 14 days after admission, wherein the line at the bottom of the plot shows the amount of MASP-2/C1-INH detected in pooled normal seronegative health care personnel, as described in example 25.

FIG. 62 is a schematic diagram showing the steps involved in a bead-based immunofluorescence assay using anti-C1 s antibodies or anti-MASP-2 antibodies immobilized on polystyrene or magnetic polystyrene microspheres (i.e., beads) to capture serine protease/C1-INH complexes (i.e., analytes) from human serum or plasma, and anti-C1-INH antibodies as detection antibodies to detect the captured complexes, as described in example 26.

FIG. 63 graphically illustrates the detection of MASP-2/C1-INH complex in pooled human serum from acute COVID-19 patients in a bead-based assay using anti-MASP-2 mAb#C8 as capture antibody, as described in example 26, as compared to BSA coated control beads.

FIG. 64 is a photograph of a non-reducing gel loaded with 6. Mu.g of a sample obtained during SEC purification of the recombinant MASP-2/C1-INH complex, as described in example 27.

FIG. 65 graphically illustrates the level of MASP-2/C1-INH complex in acute COVID-19 patients as determined in a dual bead-based assay, as described in example 28.

FIG. 66 graphically illustrates the level of C1s/C1-INH complex in acute COVID-19 patients, as determined in a dual bead-based assay, as described in example 28.

FIG. 67 graphically depicts CH of acute COVID-19 patients, convalescent patients, seropositive and seronegative persons in a longitudinal study ₅₀ Values, as described in example 28.

FIG. 68 graphically illustrates the C5a values for acute COVID-19 patients, convalescent patients, seropositive and seronegative persons in a longitudinal study, as described in example 28.

FIG. 69 graphically illustrates the levels of MASP-2/C1-INH complex in samples from 8 patients with acute COVID-19 at the time of admission (before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9-discharge) as compared to 16 healthy controls, as described in example 29.

FIG. 70A graphically illustrates CH in samples from 8 acute COVID-19 patients at admission (before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9-discharge) compared to 16 healthy controls ₅₀ Values, as described in example 29.

Figure 70B graphically illustrates the C5a values in samples from 8 acute covd-19 patients at admission (prior to naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9-discharge) as compared to 16 healthy controls, as described in example 29.

Figure 71 graphically illustrates the levels of MASP-2/C1-INH complex in samples from 7 covd-19 patients at admission (day 0, before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 2-4 after initiation of treatment; days 6-8 and 9 to discharge) compared to samples obtained from 9 covd-19 patients untreated with naloxone Li Shan anti-treatment (untreated control) and a group of healthy control subjects (healthy control) during the same time period, as described in example 30.

FIG. 72A graphically illustrates CH in samples taken from 7 COVID-19 patients at admission (day 0, before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 2-4 after initiation of treatment; days 6-8 and 9 to discharge) compared to samples taken from 9 COVID-19 patients untreated with naloxone Li Shan anti-treatment (untreated control) and a group of healthy control subjects (healthy control) during the same period of time ₅₀ Values as described in example 30.

Figure 72B graphically illustrates C5a values in samples from 7 covd-19 patients at admission (day 0, before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 2-4 after initiation of treatment; days 6-8 and 9-discharge) compared to samples obtained from 9 covd-19 patients untreated with naloxone Li Shan anti-treatment (untreated control) and a group of healthy control subjects (healthy control) during the same period of time, as described in example 30.

FIG. 73 graphically illustrates the viable bacterial count of Klebsiella pneumoniae after incubation with serum from a patient with COVID-19 prior to treatment with naloxone Li Shan (pre-treatment) and a patient with COVID-19 after treatment with naloxone Li Shan, compared to serum from a patient with COVID-19 not treated with naloxone Li Shan, compared to Normal Healthy Serum (NHS) and heat-inactivated normal healthy serum (HI-NHS), as described in example 30.

Description of sequence Listing

SEQ ID NO. 1 human MAp19 cDNA

SEQ ID NO. 2 human MAp19 protein (with leader)

SEQ ID NO. 3 human MAp19 protein (mature)

SEQ ID NO. 4 human MASP-2cDNA

SEQ ID NO. 5 human MASP-2 protein (with leader region)

SEQ ID NO. 6 human MASP-2 protein (mature)

SEQ ID NO. 7 human MASP-2 gDNA (exons 1-6)

Antigen: (reference MASP-2 mature protein)

SEQ ID NO. 8 CUBI sequence (aa 1-121)

SEQ ID NO. 9 CUBEGF sequence (aa 1-166)

SEQ ID NO:10 CUBEGFCUBII(aa 1-293)

SEQ ID NO. 11 EGF region (aa 122-166)

SEQ ID NO. 12 serine protease domain (aa 429-671)

The inactivated serine protease domain of SEQ ID NO. 13 (aa 610-625 with Ser618 to Ala mutation)

SEQ ID NO. 14 TPLGPKWPEPVFGRL (CUBI peptide)

SEQ ID NO. 15TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)

SEQ ID NO. 16 TFRSDYSN (MBL binding region core)

SEQ ID NO. 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)

SEQ ID NO. 18 IDECQVAPG (EGF peptide)

Detailed description of SEQ ID NO. 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)

Peptide inhibitors:

SEQ ID NO. 20 MBL full-length cDNA

SEQ ID NO. 21 MBL full-length protein

SEQ ID NO. 22 OGK-X-GP (consensus binding)

SEQ ID NO:23 OGKLG

SEQ ID NO:24 GLR GLQ GPO GKL GPO G

SEQ ID NO:25 GPO GPO GLR GLQ GPO GKL GPO GPO GPO

SEQ ID NO:26GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

SEQ ID NO. 27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-fiber gel protein (ficolin))

SEQ ID NO. 28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human fiber gel protein p 35)

An inhibitor of expression of SEQ ID NO 29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site):

cDNA of 30 CUBI-EGF domain (nucleotides 22-680 of SEQ ID NO: 4)

SEQ ID NO:31

5'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3'

Nucleotides 12-45 (sense) of SEQ ID NO. 4 including the MASP-2 translation initiation site

SEQ ID NO:32

5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'

Nucleotides 361-396 (sense) of SEQ ID NO. 4 encoding a region comprising a MASP-2MBL binding site

SEQ ID NO:33

5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3'

Nucleotide 610-642 clone primer of SEQ ID NO. 4 encoding a region comprising a CUBII domain:

SEQ ID NO. 34 CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for CUB)

SEQ ID NO. 35 GGAATTCCTAGGCTGCATA (3' PCR for CUB)

SEQ ID NO. 36 GGAATTCCTACAGGGCGCT (3' PCR for CUBIEGF)

SEQ ID NO. 37 GGAATTCCTAGTAGTGGAT (3' PCR for CUBIEGFCUBII)

SEQ ID NOS 38-47 is a cloning primer for a humanized antibody

SEQ ID NO. 48 is a 9aa peptide bond

Expression vector:

SEQ ID NO. 49 is a MASP-2 minigene insert

SEQ ID NO. 50 is murine MASP-2cDNA

SEQ ID NO. 51 is a murine MASP-2 protein (w/leader)

SEQ ID NO. 52 is a mature murine MASP-2 protein

SEQ ID NO. 53 rat MASP-2cDNA

SEQ ID NO. 54 is the rat MASP-2 protein (w/leader)

SEQ ID NO. 55 is a mature rat MASP-2 protein

SEQ ID NOS 56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2 for producing human MASP-2A

SEQ ID NO. 60-63 is an oligonucleotide for site-directed mutagenesis of murine MASP-2 for the production of murine MASP-2A

SEQ ID NO. 64-65 is an oligonucleotide for site-directed mutagenesis of rat MASP-2 for the production of rat MASP-2A

SEQ ID NO. 66 encoding a 17D20_dc35VH21N11VL (OMS 646) heavy chain variable region (VH) (without signal peptide)

67 17D20_dc35VH21N11VL (OMS 646) heavy chain variable region (VH) polypeptide

68 17N16mc heavy chain variable region (VH) polypeptide

69 17D20_dc35VH21N11VL (OMS 646) light chain variable region (VL) polypeptide

DNA encoding 17D20_dc35VH21N11VL (OMS 646) light chain variable region (VL) of SEQ ID NO. 70

SEQ ID NO. 71 17N16_dc17N9 light chain variable region (VL) polypeptide

SEQ ID NO. 72: SGMI-2L (full length)

SEQ ID NO. 73: SGMI-2M (moderately truncated form)

SEQ ID NO. 74: SGMI-2S (short truncated form)

SEQ ID NO. 75: mature polypeptide comprising VH-M2ab6-SGMI-2-N and human IgG4 constant region with hinge mutation

SEQ ID NO. 76: mature polypeptide comprising VH-M2ab6-SGMI-2-C and human IgG4 constant region with hinge mutation

SEQ ID NO. 77: mature polypeptide comprising VL-M2ab6-SGMI-2-N and human Ig lambda constant region

SEQ ID NO. 78: mature polypeptide comprising VL-M2ab6-SGMI-2-C and human Ig lambda constant region

SEQ ID NO. 79: peptide linker (10 aa)

SEQ ID NO. 80: peptide linker (6 aa)

SEQ ID NO. 81: peptide linker (4 aa)

SEQ ID NO. 82: polynucleotides encoding polypeptides comprising VH-M2ab6-SGMI-2-N and human IgG4 constant regions with hinge mutations

SEQ ID NO. 83: polynucleotides encoding polypeptides comprising VH-M2ab 6-SGMI-2-C and human IgG4 constant regions with hinge mutations

SEQ ID NO. 84: polynucleotides encoding polypeptides comprising VL-M2ab6-SGMI-2-N and human Ig lambda constant regions

SEQ ID NO. 85: polynucleotides encoding polypeptides comprising VL-M2ab6-SGMI-2-C and human Ig lambda constant regions

SEQ ID NO. 86: c1 inhibitor (C1-INH) homo sapiens

SEQ ID NO. 87: MASP-2 mAb C7 heavy chain variable region

SEQ ID NO. 88: MASP-2 mAb C7 light chain variable region

SEQ ID NO:89：MASP-2 mAb C7 HC-CDR1

SEQ ID NO:90：MASP-2 mAb C7 HC-CDR2

SEQ ID NO:91：MASP-2 mAb C7 HC-CDR3

SEQ ID NO:92：MASP-2 mAb C7 LC-CDR1

SEQ ID NO:93：MASP-2 mAb C7 LC-CDR2

SEQ ID NO:94：MASP-2 mAb C7 LC-CDR3

SEQ ID NO. 95: MASP-2 mAb C7 cDNA encoding the heavy chain variable region

SEQ ID NO. 96: MASP-2 mAb C7 cDNA encoding the light chain variable region

SEQ ID NO. 97: MASP-2 mAb C8 heavy chain variable region

SEQ ID NO. 98: MASP-2 mAb C8 light chain variable region

Detailed description of the preferred embodiments

As described herein, the inventors have observed that the concentration of MASP-2/C1-INH in blood (e.g., serum and/or plasma) is abnormally high in patients with severe COVID-19 as well as in subjects previously infected with COVID-19 and suffering from long-term sequelae. The inventors have also observed that after recovery, the concentration of MASP-2/C1-INH complex is in most cases reduced to normal levels. The inventors believe that it is useful to monitor the increase in MASP-2/C1-INH complex concentration in patients infected with SARS-CoV-2 for: the patient is diagnosed as having or at risk of developing acute covd-19, and the subject is also diagnosed as having or at risk of developing acute post-covd-19 (also referred to as long-covd-19), and the subject identified as having such risk is optionally treated with a complement inhibitor, e.g., a MASP-2 inhibitor. As further described herein, the use of MASP-2 inhibitors may also be useful in treating, inhibiting, reducing or preventing acute respiratory distress syndrome in a subject infected with a coronavirus, such as covd-19, and may also be useful in treating, inhibiting, reducing or preventing acute respiratory distress in a subject infected with an influenza virus. Thus, monitoring the status of the MASP-2/C1-INH complex may also be used to determine whether a patient with COVID-19 is responsive to a complement inhibitor, such as a MASP-2 inhibitor therapy, and optionally adjusting the dosage of the MASP-2 inhibitor as needed to bring the level of MASP-2/C1-INH into the normal range.

The present disclosure also provides an assay method for measuring a fluid phase MASP-2/C1-INH complex in a biological sample. Also provided are compositions, kits, and methods for interrogating a biological fluid, for example, the concentration of liquid phase MASP-2/C1-INH complex in a biological fluid obtained from a subject infected with SARS-CoV-2.

I. Definition of the definition

Unless defined otherwise herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art of the present invention. The following definitions are provided to provide clarity in describing the invention with respect to terms used in the specification and claims.

As used herein, the term "MASP-2 dependent complement activation" encompasses MASP-2 dependent activation of the lectin pathway under physiological conditions (i.e., at Ca ⁺⁺ In the presence of) results in the formation of the lectin pathway C3 convertase C4b2a and, following accumulation of the C3 cleavage product C3b, in the subsequent formation of the C5 convertase C4b2a (C3 b) n, which C4b2a (C3 b) n has been determined to cause mainly opsonization.

As used herein, the term "alternative pathway" refers to complement activation triggered, for example, by: zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from gram negative outer membranes and rabbit erythrocytes, as well as from many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and have traditionally been considered to result from spontaneous proteolytic production of C3b from complement factor C3.

As used herein, the term "lectin pathway" refers to complement activation that occurs via specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and fiber-gelling proteins (H-fiber-gelling protein, M-fiber-gelling protein, or L-fiber-gelling protein).

As used herein, the term "classical pathway" refers to complement activation that is triggered by an antibody that binds to a foreign particle and requires the binding of recognition molecule C1 q.

As used herein, the term "MASP-2 inhibitor" refers to any agent that binds to or interacts directly with MASP-2 and that is effective in inhibiting MASP-2 dependent complement activation, including anti-MASP-2 antibodies and MASP-2 binding fragments thereof, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors, and also includes peptides that compete with MASP-2 for binding to another recognition molecule in the lectin pathway (e.g., MBL, H-fiber-gelling protein, M-fiber-gelling protein, or L-fiber-gelling protein), but does not include antibodies that bind to such other recognition molecules. MASP-2 inhibitors useful in the methods of the invention may reduce MASP-2 dependent complement activation by greater than 20%, such as greater than 50%, such as greater than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2 dependent complement activation by greater than 90% (i.e., results in only 10% or less of MASP-2 complement activation).

As used herein, the term "fibrosis" refers to the formation or presence of excess connective tissue in an organ or tissue. Fibrosis may occur as a repair or replacement response to a stimulus such as tissue injury or inflammation. The hallmark of fibrosis is the production of excessive extracellular matrix. Normal physiological responses to injury result in connective tissue deposition as part of the healing process, but such connective tissue deposition may persist and become pathological, altering the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia.

As used herein, the term "treating fibrosis in a mammalian subject suffering from or at risk of developing a disease or condition caused by or exacerbated by fibrosis and/or inflammation" refers to reversing, alleviating, ameliorating or inhibiting fibrosis in the mammalian subject.

As used herein, the term "proteinuria" refers to the presence of urinary protein in abnormal amounts, e.g., in an amount of more than 0.3g protein in a 24 hour urine collection from a human subject, or at a concentration of more than 1 g/liter in a human subject.

As used herein, the term "ameliorating proteinuria" or "reducing proteinuria" refers to reducing 24-hour urinary protein excretion in a subject suffering from proteinuria by at least 20%, such as at least 30%, such as at least 40%, such as at least 50% or more, as compared to the baseline 24-hour urinary protein excretion in the subject prior to treatment with a MASP-2 inhibitor. In one embodiment, treatment with a MASP-2 inhibitor according to the methods of the invention is effective to reduce proteinuria in a human subject such that greater than 20 percent reduction in 24 hours urine protein excretion, or greater than 30 percent reduction in, for example, 24 hours urine protein excretion, or greater than 40 percent reduction in, for example, 24 hours urine protein excretion, or greater than 50 percent reduction in, for example, 24 hours urine protein excretion is achieved.

As used herein, the terms "small molecule," "small organic molecule," and "small inorganic molecule" refer to molecules (organic, organometallic, or inorganic), organic molecules, and inorganic molecules, respectively, that are naturally occurring or synthetic, and have a molecular weight of greater than about 50Da and less than about 2500 Da. The small organic (e.g.,) molecules may be less than about 2000Da, between about 100Da and about 1000Da, or between about 100 and about 600Da, or between about 200 and 500 Da.

As used herein, the term "antibody" includes antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mice, rats, rabbits, and primates including humans), or hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments), andspecifically binds to a target polypeptide, such as MASP-2, a polypeptide or a portion thereof. The term "antibody" is not intended to be limited in the source of the antibody, or the manner in which it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); a humanized antibody; a murine antibody; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, fab, fab ', F (ab') ₂ Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion comprising an antigen binding fragment of the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen binding site or fragment (epitope recognition site) of the desired specificity.

"monoclonal antibody" refers to a homogeneous population of antibodies, wherein the monoclonal antibodies comprise amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific for the target antigen. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full length monoclonal antibodies, but also fragments thereof (e.g., fab ', F (ab') ₂ Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen binding fragment (epitope recognition site) having the desired specificity and ability to bind an epitope. It is not intended to be limited in the source of the antibody, or the manner in which it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes intact immunoglobulins and the like as described above in "antibodies Fragments and the like described below are defined.

As used herein, the term "antibody fragment" refers to a portion derived from or associated with a full length antibody, such as an anti-MASP-2 antibody, generally including the antigen-binding or variable regions thereof. Illustrative examples of antibody fragments include Fab, fab ', F (ab) 2, F (ab') 2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, a "single chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

As used herein, a "chimeric antibody" is a recombinant protein that contains variable domains and complementarity determining regions derived from antibodies of a non-human species (e.g., rodents), while the remainder of the antibody molecule is derived from a human antibody.

As used herein, a "humanized antibody" is a chimeric antibody that comprises minimal sequences consistent with specific complementarity determining regions derived from non-human immunoglobulins that are grafted into human antibody frameworks. Humanized antibodies are typically recombinant proteins in which only the complementarity determining regions of the antibody are of non-human origin.

As used herein, the term "mannan-binding lectin" ("MBL") is equivalent to the mannan-binding protein ("MBP").

As used herein, a "membrane attack complex" ("MAC") refers to a complex of five complement components (C5 b in combination with C6, C7, C8, and C-9) (also referred to as C5 b-9) that intercalates into the membrane and destroys the end of the membrane.

As used herein, "subject" includes all mammals including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

Naturally occurring amino acids in the broadest sense can be divided into groups based on the chemical characteristics of the side chains of the respective amino acids. "hydrophobic" amino acid means Ile, leu, met, phe, trp, tyr, val, ala, cys or Pro. By "hydrophilic" amino acid is meant Gly, asn, gln, ser, thr, asp, glu, lys, arg or His. This grouping of amino acids can be further reclassified as follows. "uncharged hydrophilic" amino acids mean Ser, thr, asn or Gln. "acidic" amino acid means Glu or Asp. "basic" amino acid means Lys, arg or His.

As used herein, the term "conservative amino acid substitutions" is illustrated by the substitution of amino acids within each of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimetic thereof. The term also encompasses oligonucleotide bases (oligonucleobases) consisting of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring modifications.

As used herein, an "epitope" refers to a site on a protein (e.g., human MASP-2 protein) that is bound by an antibody. "overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residue, including linear epitopes and non-linear epitopes.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. The MASP-2 proteins described herein may contain or may be wild-type proteins, or may be variants having no more than 50 (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; phenylalanine and tyrosine.

In some embodiments, the human MASP-2 protein may have an amino acid sequence that is equal to or greater than 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100)% identical to the human MASP-2 protein, which has the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the peptide fragment may be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues in length (e.g., at least 6 contiguous amino acid residues of SEQ ID NO: 5). In some embodiments, the antigenic peptide fragment of a human MASP-2 protein is less than 500 (e.g., less than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length (e.g., less than 500 contiguous amino acid residues in any of SEQ ID NO: 5).

The percent (%) amino acid sequence identity is defined as: after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, the percentage of amino acids in the candidate sequence that are identical to the amino acids in the reference sequence. Alignment for the purpose of determining percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. Suitable parameters for measuring the alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the sequences to be compared.

Summary of the invention

As described herein, the inventors have identified a central role for the lectin pathway in the initiation of renal tubular kidney disease conditions and disease progression, suggesting a critical role for lectin pathway activation in the pathophysiology of a diverse range of kidney diseases including IgA nephropathy, C3 glomerulopathy and other glomerulonephritis. As further described herein, the inventors have discovered that inhibition of the key regulator of the lectin pathway of the complement system, namely mannan-binding lectin associated serine protease-2 (MASP-2), significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including Unilateral Ureteral Obstruction (UUO) models, protein overload models, and doxorubicin-induced kidney fibrotic nephropathy models. Thus, the inventors have demonstrated that inhibition of MASP-2 mediated lectin pathway activation provides an effective therapeutic approach to improve, treat or prevent renal fibrosis, e.g., tubular interstitial fibrosis, regardless of the underlying cause. As further described herein, the use of MASP-2 inhibitors may also be useful in treating, inhibiting, alleviating or preventing acute respiratory distress syndrome in a subject infected with a coronavirus, such as covd-19.

Lectins (MBL, M-fiber-gel protein, H-fiber-gel protein, L-fiber-gel protein, and CL-11) are specific recognition molecules that trigger the congenital complement system, and the system includes lectin-initiated pathways and associated terminal pathways amplifying loops that amplify lectin-initiated activation of terminal complement effector molecules. C1q is a specific recognition molecule that triggers the acquired complement system, and the system includes a classical initiation pathway and an associated terminal pathway amplification loop that amplifies activation of the C1 q-initiated terminal complement effector molecule. We refer to these two major complement activation systems as the lectin-dependent complement system and the C1 q-dependent complement system, respectively.

In addition to its essential role in immune defenses, the complement system contributes to tissue damage in many clinical situations. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. It is recognized that lectin-mediated MASP-2 pathway can be inhibited while leaving the classical pathway intact, and thus it is highly desirable to specifically inhibit only the complement activation system that causes a particular pathological condition, without completely shutting down the immune defenses of complement. For example, in disease states in which complement activation is predominantly mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This leaves the C1 q-dependent complement activation system intact to handle immune complex processing and to aid host defense against infection.

A preferred protein component targeted to specifically inhibit the lectin-dependent complement system in the development of therapeutic agents is MASP-2. Of all known protein components of the lectin-dependent complement system (MBL, H-fiber gelonin, M-fiber gelonin, L-fiber gelonin, MASP-2, C2-C9, factor B, factor D and properdin), only MASP-2 is both unique to the lectin-dependent complement system and necessary for the system to function. Lectins (MBL, H-fiber gelator, M-fiber gelator, L-fiber gelator, and CL-11) are also unique components in the lectin-dependent complement system. However, due to lectin redundancy, the loss of any one lectin component does not necessarily inhibit activation of the system. It is necessary to inhibit all five lectins in order to ensure inhibition of the lectin-dependent complement activation system. Furthermore, since MBL and fibrin are also known to have complement independent opsonic activity, inhibition of lectin function will result in the loss of this beneficial host defense mechanism against infection. In contrast, if MASP-2 is the inhibitory target, this complement-independent lectin opsonic activity will remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibition of the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is the lowest of any complement proteins (+.about.500 ng/ml); accordingly, a correspondingly low concentration of a high affinity inhibitor of MASP-2 may be sufficient to obtain complete inhibition (Moller-Kristensen, M.et al, J.Immunol Methods 282:159-167, 2003).

As described in example 14 herein, mice that do not contain the MASP-2 gene (MASP-2-/-) exhibited significantly less kidney disease as compared to wild-type control animals as determined in animal models of fibrotic kidney disease (unilateral ureteral obstruction UUO), as indicated by inflammatory cell infiltration (75% decrease) and histological markers of fibrosis as collagen deposition (one third decrease). As further shown in example 15, wild-type mice treated with the anti-MASP-2 monoclonal antibody system, which selectively blocks the lectin pathway while leaving the classical pathway intact, were protected from renal fibrosis compared to wild-type mice treated with isotype control antibodies. These results demonstrate that the lectin pathway is a key contributor to kidney disease and further demonstrate that MASP-2 inhibitors (e.g., MASP-2 antibodies) that block the lectin pathway are effective as anti-fibrotic agents. As further shown in example 16, wild-type mice treated with Bovine Serum Albumin (BSA) developed proteinuria nephropathy in a protein overload model, while MASP-2-/-mice treated with the same level of BSA had reduced kidney injury. As shown in example 17, wild-type mice treated with an anti-MASP-2 monoclonal antibody system that selectively blocks the lectin pathway while leaving the classical pathway intact were protected from kidney injury in a protein overload model. As described in example 18, MASP-2-/-mice showed less kidney inflammation and tubular interstitial damage in the doxorubicin-induced kidney fibrosis kidney disease model compared to wild-type mice. In the ongoing phase 2 open-labeled kidney trial, igA nephropathy patients treated with anti-MASP-2 antibodies demonstrated clinically significant and statistically significant reductions in urinary albumin/creatinine ratio (uACR) throughout the trial, as well as reductions in urine protein levels at 24 hours from baseline to the end of treatment, as described in example 19. As further described in example 19, membranous nephropathy patients treated with anti-MASP-2 antibodies also demonstrated a decrease in uACR during treatment in the same phase 2 kidney assay.

In accordance with the foregoing, the present invention relates to the use of a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, as an anti-fibrotic agent, the use of a MASP-2 inhibitor for the manufacture of a medicament for the treatment of a fibrotic condition, and a method of preventing, treating, alleviating or reversing a fibrotic condition in a human subject in need thereof, the method comprising administering to the patient an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody). As described in examples 20, 21 and 22, clinical improvement was observed in patients with covd-19 associated respiratory failure following anti-treatment with nano-wire Li Shan, which anti-inhibits MASP-2 and lectin pathway activation, nano-wire Li Shan. All 6 patients with covd-19 treated with naloxone Li Shan demonstrated clinical improvement as described in example 21. In each case, covd-19 lung injury had progressed to ARDS before nano-cable Li Shan anti-treatment, and all patients received non-invasive mechanical ventilation at the initiation of treatment. The patient with COVID-19, whose follow-up (5-6 months) data was available for treatment with naloxone Li Shan, did not show clinical or laboratory evidence of long-term sequelae observed. As further described in example 22, additional COVID-19 patients treated with naloxone Li Shan resistance also demonstrated clinical improvement. As further demonstrated in example 22, patients treated with the naloxone Li Shan antibody developed appropriately high titers of anti-SARS-Cov-2 antibodies, indicating that treatment with the naloxone Li Shan antibody did not block the effector function of the adaptive immune response.

As further described in examples 24-30 herein, the inventors have observed that the concentration of MASP-2/C1-INH in blood (e.g., serum and/or plasma) is abnormally high in patients with severe covd-19 as well as in subjects previously infected with covd-19 and suffering from long-term sequelae. The inventors have also observed that after recovery, the concentration of MASP-2/C1-INH complex is in most cases reduced to normal levels. The inventors believe that it is useful to monitor the increase in MASP-2/C1-INH complex concentration in patients infected with SARS-CoV-2 for: the patient is diagnosed as having or at risk of developing acute covd-19, and the subject is also diagnosed as having or at risk of developing acute post-covd-19 (also referred to as long-covd-19), and the subject identified as having such risk is optionally treated with a complement inhibitor, e.g., a MASP-2 inhibitor. As further described herein, the use of MASP-2 inhibitors may also be useful in treating, inhibiting, reducing or preventing acute respiratory distress syndrome in a subject infected with a coronavirus, such as covd-19, and may also be useful in treating, inhibiting, reducing or preventing acute respiratory distress in a subject infected with an influenza virus. Thus, monitoring the status of the MASP-2/C1-INH complex may also be used to determine whether a patient with COVID-19 is responsive to a complement inhibitor, such as a MASP-2 inhibitor therapy, and optionally adjusting the dosage of the MASP-2 inhibitor as needed to bring the level of MASP-2/C1-INH into the normal range.

Thus, the methods of the invention may be used to treat, inhibit, reduce, prevent or reverse coronavirus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from a coronavirus, such as covd-19, SARS or MERS, as further described herein. The methods of the invention may also be used to treat, inhibit, alleviate, prevent or reverse influenza virus-induced pneumonia or acute respiratory distress syndrome in a human subject suffering from influenza virus, such as influenza a serotypes (H1N 1 (causing "spanish influenza" in 1918 and "swine influenza" in 2009 "); H2N2 (causing" asian influenza "in 1957), H3N2 (causing" hong kong influenza "in 1968), H5N1 (causing" avian influenza "in 2004), H7N7, H1N2, H9N2, H7N3, H10N7, H7N9 and H6N 1); or influenza b virus or influenza c virus.

III. roles of MASP-2 in diseases and conditions caused or exacerbated by fibrosis

Fibrosis is the excessive connective tissue formation or presence in an organ or tissue, typically in response to injury or damage. The hallmark of fibrosis is the production of excessive extracellular matrix following injury. In the kidneys, fibrosis is characterized by progressive, deleterious connective tissue deposition in the kidney parenchyma, which inevitably leads to reduced renal function, independent of the primary renal disease that caused the original kidney injury. So-called Epithelial Mesenchymal Transition (EMT), in which tubular epithelial cells are transformed into mesenchymal fibroblasts, a change in cellular characteristics constitutes the primary mechanism of renal fibrosis. Fibrosis affects almost all tissue and organ systems and may occur as a repair or replacement response to a stimulus, such as tissue injury or inflammation. Normal physiological responses to injury result in deposition of connective tissue, but if this process becomes pathological, the highly differentiated cells alter the architecture and function of the tissue through the replacement of scarring connective tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia. There is no effective treatment or therapeutic for fibrosis at present, but both animal studies and biogenic human reports suggest that fibrotic tissue damage can be reversed (Tampe and Zeisberg, nat Rev Nephrol, vol. 10: 226-237, 2014).

Many diseases lead to fibrosis that causes progressive organ failure, including the following: kidneys (e.g., chronic kidney disease, igA nephropathy, C3 glomerulonephritis and other glomerulonephritis), lungs (e.g., idiopathic pulmonary fibrosis, cystic fibrosis, bronchodilation), liver (e.g., liver cirrhosis, nonalcoholic fatty liver disease), heart (e.g., myocardial infarction, atrial fibrosis, valve fibrosis, endocardial fibrosis), brain (e.g., stroke), skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, keloids), vascular system (e.g., atherosclerotic vascular disease), intestinal tract (e.g., crohn's disease), eye (e.g., subcapsular cataract, posterior capsule turbidity), musculoskeletal soft tissue structure (e.g., adhesive joint capsulitis, dupuytren's contracture), myelofibrosis), reproductive organs (e.g., endometriosis, pecies disease), and certain infectious diseases (e.g., coronaviruses, alphaviruses, hepatitis C, hepatitis b, etc.).

Although fibrosis occurs in many tissues and diseases, there are common molecular and cellular mechanisms of their pathological conditions. Extracellular matrix deposition by fibroblasts is accompanied by immune cell infiltration, predominantly monocytes (see Wynn t., nat Rev Immunol4 (8): 583-594, 2004, incorporated herein by reference). The robust inflammatory response results in the expression of: growth factors (TGF-beta, VEGF, hepatocyte growth factor, connective tissue growth factor), cytokines and hormones (endothelin, IL-4, IL-6, IL-13, chemokines), degrading enzymes (elastase, matrix metalloproteinases, cathepsins) and extracellular matrix proteins (collagen, fibronectin, integrins).

In addition, the complement system becomes activated in numerous fibrotic diseases. Complement components, including membrane attack complexes, have been identified in numerous fibrotic tissue samples. For example, components of the lectin pathway have been found in the following fibrotic lesions: kidney disease (Satomura et al, nephron.92 (3): 702-4 (2002); sato et al, lupus 20 (13): 1378-86 (2011); liu et al, clin Exp Immunol,174 (1): 152-60 (2013)); liver disease (Rensen et al, hepatology 50 (6): 1809-17 (2009)); and pulmonary diseases (Olesen et al, clin Immunol 121 (3): 324-31 (2006)).

Upregulated complement activation has been identified as a key contributor to immune complex mediated and antibody independent glomerulonephritis. However, there is a strong chain of evidence that in situ uncontrolled complement activation is essentially involved in the pathophysiological progression of TI fibrosis in non-glomerular disease (Quigg R.J, J Immunol 171:3319-3324, 2003, naik A. Et al, semin Nephrol 33:575-585, 2013, matsouthern D.R. et al, clin J Am Soc Nephrol10:P1636-1650, 2015). The strong pro-inflammatory signal triggered by local complement activation can be initiated by: complement components that filter into the proximal tubule and subsequently enter the interstitial space, or abnormal synthesis of complement components by the renal tubule or other resident and infiltrating cells, or abnormal expression of complement regulatory proteins on kidney cells, or absence or loss or acquisition of functional mutations in complement regulatory components (Mather D.R. et al Clin J Am Soc Nephrol10:P1636-1650, 2015, shearin N.S. et al FASEB J22:1065-1072, 2008). For example, in mice, a deficiency in complement regulator protein CR 1-associated gene/protein y (Crry) results in Tubulointerstitial (TI) complement activation, with subsequent inflammation and impairment typical of fibrosis seen in human TI disease (Naik A. Et al, semin Nephrol 33:575-585, 2013, bao L. Et al, JAm Soc Nephrol 18:811-822, 2007). Exposure of tubular epithelial cells to anaphylatoxin C3a results in epithelial mesenchymal transition (Tsang Z. Et al, J Am Soc Nephrol 20:593-603, 2009). Blocking C3a signaling via the C3a receptor alone has recently been shown to reduce Kidney TI fibrosis in proteinuria and non-proteinuria animals (Tsang Z. Et al, J Am Soc Nephrol 20:593-603, 2009, bao L. Et al, kidney int.80:524-534, 2011).

As described herein, the inventors have identified a central role for the lectin pathway in the initiation of renal tubular renal disease conditions and in disease progression, suggesting a critical role for lectin pathway activation in the pathophysiology of a diverse range of renal diseases including IgA nephropathy, C3 glomerulopathy and other glomerulonephritis (Endo M.et al, nephrol Dialysis Transplant:1984-1990, 1998; hisano S. Et al, am J Kidney Dis 45:295-302, 2005; roos a. Et al, J Am Soc Nephrol 17:1724-1734, 2006; liu l.l. Et al, clin exp. Immunol 174:152-160, 2013; lhotta K. Et al, nephrol Dialysis Transplant:881-886, 1999; pickering et al, kidney International:1079-1089, 2013), diabetic nephropathy (Hovind p. Et al, diabetes 54:1523-1527, 2005), ischemic reperfusion injury (Asgari e. Et al, FASEB J28:3996-4003, 2014) and graft rejection (Berger s.p. et al, am J Transplant 5:1361-1366, 2005).

As further described herein, the inventors have demonstrated that MASP-2 inhibition reduces inflammation and fibrosis in a mouse model of tubular interstitial disease. Thus, inhibitors of MASP-2 are expected to be useful in the treatment of renal fibrosis, including tubular interstitial inflammation and fibrosis, proteinuria, igA nephropathy, C3 glomerulopathy and other glomerulonephritis and renal ischemia reperfusion injury.

Pulmonary disease

Pulmonary fibrosis is the formation or development of excessive fibrous connective tissue in the lungs, where normal lung tissue is replaced by fibrotic tissue. This scarring results in stiffness in the lungs and impairs lung structure and function. In humans, pulmonary fibrosis is thought to result from repeated damage to tissues within and between tiny air sacs (alveoli) in the lungs. Under experimental settings, various animal models have replicated aspects of human disease. For example, an external agent such as bleomycin, fluorescein isothiocyanate, silica or asbestos may be instilled into the trachea of the animal (Gharaee-kerani et al, animal Models of Pulmonary fibris. Methods mol. Med.,2005, 117:251-259).

Thus, in certain embodiments, the present disclosure provides methods of inhibiting pulmonary fibrosis in a subject having a pulmonary disease or disorder caused by or exacerbated by fibrosis and/or inflammation, such as coronavirus-induced ARDS, comprising administering a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, to a subject in need thereof. The method comprises administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit pulmonary fibrosis, reduce pulmonary fibrosis, and/or improve pulmonary function. Improvement of symptoms of lung function includes improvement of lung function and/or volume, reduction of fatigue, and improvement of oxygen saturation.

The MASP-2 inhibitory composition may be topically applied to the fibrotic area, for example, during surgery or local injection, by applying the composition locally, either directly or remotely (e.g., via a catheter). Alternatively, the MASP-2 inhibitor may be administered to the subject systemically, e.g., by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration with respect to the non-peptide energetic agent. Administration may be repeated as determined by the physician until the condition has resolved or is controlled.

In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more agents or therapeutic modalities appropriate for the underlying pulmonary disease or condition.

Infectious disease

Infectious diseases such as coronaviruses and chronic infectious diseases such as hepatitis c and hepatitis b cause tissue inflammation and fibrosis, and high lectin pathway activity may be detrimental. Inhibitors of MASP-2 may be beneficial in such diseases. For example, MBL and MASP-1 levels were found to be important predictors of liver fibrosis severity in Hepatitis C Virus (HCV) infection (Brown et al, clin Exp immunol.147 (1): 90-8, 2007; saadanay et al, arab J gastroenterol.12 (2): 68-73, 2011; saeed et al, clin Exp immunol.174 (2): 265-73, 2013). MASP-1 has previously been shown to be a powerful activator of the MASP-2 and lectin pathways (Megyeri et al, J Biol chem.29:288 (13): 8922-34, 2013). Alphaviruses such as chikungunya and ross river induce strong host inflammatory responses leading to arthritis and myositis, and this pathological condition is mediated by MBL and lectin pathways (Gunn et al, PLoS pathg.8 (3): e1002586, 2012).

Thus, in certain embodiments, the present disclosure provides methods of preventing, treating, reversing, inhibiting and/or reducing fibrosis and/or inflammation in a subject having or having previously had an infectious disease that causes inflammation and/or fibrosis, such as a coronavirus or an influenza virus, comprising administering a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, to a subject in need thereof.

In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered in combination with one or more agents or therapeutic modalities appropriate for the underlying infectious disease.

In some embodiments, the infectious disease that causes inflammation and/or fibrosis is selected from the group consisting of coronavirus, alphavirus, hepatitis a, hepatitis b, hepatitis c, tuberculosis, HIV, and influenza.

In certain embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a MASP-2 inhibitory small molecule compound) is administered in combination with one or more agents or therapeutic modalities appropriate for the underlying disease or disorder.

In certain embodiments of any of the various methods and pharmaceutical compositions described herein, the MASP-2 inhibitory antibody or small molecule compound selectively blocks the lectin pathway while leaving the classical pathway intact.

MASP-2 inhibitors

In various aspects, the invention provides methods of inhibiting the adverse effects of fibrosis and/or inflammation comprising administering a MASP-2 inhibitor to a subject in need thereof. The MASP-2 inhibitor is administered in an amount effective to inhibit MASP-2 dependent complement activation in a living subject. In the practice of this aspect of the invention, representative MASP-2 inhibitors include: molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, anti-MASP-2 antibodies (e.g., MASP-2 inhibitory antibodies), or blocking peptides that interact with MASP-2 or interfere with protein-protein interactions), and molecules that reduce MASP-2 expression (e.g., MASP-2 antisense nucleic acid molecules, MASP-2 specific RNAi molecules, and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complement pathway. MASP-2 inhibitors may be used as primary therapies alone or in combination with other therapeutic agents as adjuvant therapies to enhance the therapeutic benefits of other medical therapies.

Inhibition of MASP-2 dependent complement activation is characterized by at least one of the following changes in the complement system components that occur as a result of administration of a MASP-2 inhibitor according to the methods of the invention: inhibition of the production or production of MASP-2 dependent complement activation system products C4b, C3a, C5a, and/or C5b-9 (MAC) (e.g., as measured in example 2), reduction of C4 cleavage and C4b deposition (e.g., as measured in example 2), or reduction of C3 cleavage and C3b deposition (e.g., as measured in example 2).

According to the present invention, MASP-2 inhibitors are utilized which are effective in inhibiting respiratory distress (or in other words, improving respiratory function) in a subject infected with coronavirus.

The evaluation of respiratory function may be performed periodically, for example, hourly, daily, weekly or monthly. Such evaluation is preferably performed at several time points for a given subject or at one or several time points for a given subject and a healthy control. The evaluation may be performed at regular intervals, for example, hourly, daily, weekly or monthly. When an assessment has resulted in the discovery that respiratory distress is reduced (i.e., respiratory function is increased), MASP-2 inhibitors, such as MASP-2 inhibitory antibodies, are said to be effective in treating subjects with coronavirus-induced acute respiratory distress syndrome.

MASP-2 inhibitors useful in the practice of this aspect of the invention include, for example, MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. MASP-2 inhibitors may inhibit the MASP-2 dependent complement activation system by blocking the biological function of MASP-2. For example, inhibitors may be effective in blocking MASP-2 protein-protein interactions, interfering with MASP-2 dimerization or assembly, blocking Ca ²⁺ Binding, interfering with the MASP-2 serine protease active site, or possibly reducing MASP-2 protein expression.

In some embodiments, the MASP-2 inhibitor selectively inhibits MASP-2 complement activation, leaving the C1 q-dependent complement activation system functionally intact.

In one embodiment, the MASP-2 inhibitors useful in the methods of the invention are specific MASP-2 inhibitors that specifically bind to a polypeptide comprising SEQ ID NO. 6, which have at least ten times the affinity to other antigens in the complement system. In another embodiment, the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO. 6 with an affinity of at least 100-fold to other antigens in the complement system. In one embodiment, the MASP-2 inhibitor specifically binds to at least one of the following: (i) CCP1-CCP2 domain (aa 300-431 of SEQ ID No. 6) or serine protease domain of MASP-2 (aa 445-682 of SEQ ID No. 6) and inhibits MASP-2 dependent complement activation. In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to MASP-2. Binding affinity of MASP-2 inhibitors may be determined using a suitable binding assay.

MASP-2 polypeptides exhibit molecular structures similar to MASP-1, MASP-3, and C1r and C1s (proteases of the C1 complement system). The cDNA molecule shown in SEQ ID NO. 4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO. 5) and provides a human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion resulting in the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIG. 2, the human MASP 2 gene comprises twelve exons. Human MASP-2cDNA is encoded by exons B, C, D, F, G, H, I, J, K and L. Alternative splicing results in a 20kDa protein, called MBL-associated protein 19 ("MAp 19", also called "sMAP") (SEQ ID NO: 2), encoded by (SEQ ID NO: 1), originating from exons B, C, D and E, as shown in FIG. 2. The cDNA molecule shown in SEQ ID NO. 50 encodes murine MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO. 51) and provides a murine MASP-2 polypeptide with a leader sequence that is cleaved after secretion resulting in the mature form of murine MASP-2 (SEQ ID NO. 52). The cDNA molecule shown in SEQ ID NO. 53 encodes rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO. 54) and provides the rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion resulting in the mature form of rat MASP-2 (SEQ ID NO: 55).

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO. 4, SEQ ID NO. 50 and SEQ ID NO. 53 represent individual alleles of human, murine and rat MASP-2, respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID No. 4, SEQ ID No. 50 and SEQ ID No. 53, including allelic variants comprising silent mutations, and allelic variants wherein the mutation results in an amino acid sequence change, are within the scope of the invention. Allelic variants of MASP-2 sequences may be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

The domains of the human MASP-2 protein (SEQ ID NO: 6) are shown in FIGS. 1 and 2A and include the N-terminal C1r/C1 s/sea urchin Vegf/bone morphogenic protein (CUBI) domain (aa 1-121 of SEQ ID NO: 6), the epidermal growth factor-like domain (aa 122-166), the second CUBI domain (aa 167-293), and the tandem complement control protein domain and serine protease domain. Alternative splicing of the MASP 2 gene results in MAP19 shown in FIG. 1. MAp19 is a non-enzymatic protein that contains the N-terminal CUBI-EGF region of MASP-2, accompanied by four additional residues (EQSL) derived from exon E, as shown in FIG. 1.

Several proteins have been shown to bind or interact with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind to and form Ca with lectin proteins MBL, H-fiber gelata and L-fiber gelata ²⁺ A dependent complex. Each MASP-2/lectin complex has been shown to activate complement by MASP-2 dependent cleavage of proteins C4 and C2 (Ikeda, K. Et al, J.biol. Chem.262:7451-7454, 1987; matsushita, M. Et al, J.exp. Med.176:1497-2284, 2000; matsushita, M. Et al, J.Immunol.168:3502-3506, 2002). Studies have shown that the CUBI-EGF domain of MASP-2 is necessary for binding of MASP-2 to MBL (Thielens, N.M. et al, J.Immunol.166:5068, 2001). The CUBIEGFCUBII domain has also been shown to mediate dimerization of MASP-2, which is necessary for the formation of active MBL complexes (Wallis, R. Et alHuman, J.biol. Chem.275:30962-30969, 2000). Thus, MASP-2 inhibitors may be identified that bind to or interfere with MASP-2 target regions known to be important for MASP-2 dependent complement activation.

anti-MASP-2 inhibitory antibodies

In some embodiments of this aspect of the invention, the MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits a MASP-2 dependent complement activation system. anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal, or recombinant antibodies derived from any antibody-producing mammal, and may be multispecific, chimeric, humanized, anti-idiotype, and antibody fragments. Antibody fragments include Fab, fab ', F (ab) 2, F (ab') 2, fv fragments, scFv fragments, and single chain antibodies as further described herein.

Using the assays described herein, MASP-2 antibodies may be screened for their ability to inhibit the ability and anti-fibrotic activity of the MASP-2 dependent complement activation system, and/or for their ability to inhibit kidney damage associated with proteinuria or doxorubicin-induced kidney disease. Several MASP-2 antibodies have been described in the literature and some have been newly generated, some of which are listed in Table 1 below. For example, anti-MASP-2 Fab2 antibodies that block MASP-2 dependent complement activation have been identified as described in examples 10 and 11 herein. Fully human MASP-2scFv antibodies (e.g., OMS 646) that block MASP-2 dependent complement activation have been identified as described in example 12 and in WO2012/151481, which is incorporated herein by reference. SGMI-2 peptide-loaded MASP-2 antibodies and fragments thereof having MASP-2 inhibitory activity are produced by fusing an SGMI-2 peptide amino acid sequence (SEQ ID NO:72, 73 or 74) to the amino or carboxy terminus of the heavy and/or light chain of a human MASP-2 antibody (e.g., OMS 646-SGMI-2) as described in example 13 and in WO2014/144542, which is incorporated herein by reference.

Thus, in one embodiment, the MASP-2 inhibitor used in the methods of the invention comprises a human antibody, such as OMS646. Thus, in one embodiment, the MASP-2 inhibitor used in the compositions and methods of the invention comprises a human antibody that binds to a polypeptide consisting of human MASP-2 (SEQ ID NO: 6), wherein the antibody comprises: (I) (a) a heavy chain variable region comprising: i) A heavy chain CDR-H1 comprising an amino acid sequence from 31 to 35 of SEQ ID NO. 67; and ii) a heavy chain CDR-H2 comprising an amino acid sequence from 50 to 65 of SEQ ID NO. 67; and iii) a heavy chain CDR-H3 comprising an amino acid sequence from 95 to 107 of SEQ ID NO. 67, and b) a light chain variable region comprising: i) Light chain CDR-L1 comprising the amino acid sequence of 24-34 from SEQ ID NO. 69; and ii) light chain CDR-L2 comprising the amino acid sequence from 50 to 56 of SEQ ID NO. 69; and iii) a light chain CDR-L3 comprising an amino acid sequence from 89-97 of SEQ ID NO. 69, or (II) a variant thereof comprising a heavy chain variable region having 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO. 67, and a light chain variable region having 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO. 69.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen binding fragment thereof, said MASP-2 inhibitory antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 67, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69.

In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 that is recognized by reference antibody OMS646, which reference antibody OMS646 comprises a heavy chain variable region as set forth in SEQ ID NO:67 and a light chain variable region as set forth in SEQ ID NO: 69. In one embodiment, the MASP-2 inhibitor used in the methods of the invention comprises human antibody OMS646.

Table 1: exemplary MASP-2-specific antibodies

anti-MASP-2 antibodies with reduced effector function

In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies have reduced effector function in order to reduce inflammation that may result from activation of the classical complement pathway. The ability of IgG molecules to trigger the classical complement pathway has been shown to be located within the Fc portion of the molecule (Duncan, A.R. et al, nature 332:738-740 1988). IgG molecules from which the Fc portion of the molecule has been removed by enzymatic cleavage lack this effector function (see Harlow, antibodies: A Laboratory Manual, cold Spring Harbor Laboratory, new York, 1988). Thus, by having a genetically engineered Fc sequence that minimizes effector function, or having a human IgG2 or IgG4 isotype, antibodies with reduced effector function are produced due to the lack of the Fc portion of the molecule.

Antibodies with reduced effector function can be produced by standard molecular biological manipulations of the Fc portion of the IgG heavy chain as described herein and in Jolliffe et al, int' l Rev. Immunol.10:241-250, 1993, and Rodrigues et al, J.Immunol.151:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that have reduced ability to activate complement and/or interact with Fc receptors (Ravetch, J.V. et al, annu.Rev. Immunol.9:457-492, 1991; isaacs, J.D. et al, J.Immunol.148:3062-3071, 1992;van de Winkel,J.G. Et al, immunol.today 14:215-221, 1993). Humanized or fully human antibodies specific for human MASP-2 consisting of the IgG2 or IgG4 isotype can be produced by one of several methods known to one of ordinary skill in the art, as described in Vaughan, T.J. et al, nature Biotechnical 16:535-539, 1998.

Production of anti-MASP-2 antibodies

anti-MASP-2 antibodies may be produced using a MASP-2 polypeptide (e.g., full length MASP-2), or using a peptide bearing an antigenic MASP-2 epitope (e.g., a portion of a MASP-2 polypeptide). Immunogenic peptides can be as small as five amino acid residues. For example, MASP-2 polypeptides comprising the entire amino acid sequence of SEQ ID NO. 6 may be used to induce anti-MASP-2 antibodies that may be used in the methods of the invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as regions comprising serine protease active sites, can be expressed as recombinant polypeptides and used as antigens as described in example 3. In addition, peptides comprising at least a portion of 6 amino acids of the MASP-2 polypeptide (SEQ ID NO: 6) may also be used to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens that may be used to induce MASP-2 antibodies are provided in Table 2 below. MASP-2 peptides and polypeptides for producing antibodies may be isolated as natural polypeptides, or recombinant or synthetic peptides, or non-catalytically active recombinant polypeptides such as MASP-2A, as further described herein. In some embodiments of this aspect of the invention, transgenic mouse strains as described herein are used to obtain anti-MASP-2 antibodies.

Antigens useful in the production of anti-MASP-2 antibodies also include fusion polypeptides, such as fusion of MASP-2 or a portion thereof with an immunoglobulin polypeptide or maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide moiety is hapten-like, such moiety may be advantageously conjugated or linked to a macromolecular carrier, such as Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA) or tetanus toxoid, for immunization.

Table 2: MASP-2 derived antigens

Polyclonal antibodies

Polyclonal antibodies to MASP-2 may be prepared by immunizing an animal with a MASP-2 polypeptide or immunogenic portions thereof using methods well known to those of ordinary skill in the art. See, e.g., green et al, "Production of Polyclonal Antisera," at Immunochemical Protocols (Manson, eds.), page 105. Immunogenicity of MASP-2 polypeptides may be increased by the use of adjuvants including mineral gels, such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are typically produced in animals such as horses, cattle, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, anti-MASP-2 antibodies useful in the invention may also be derived from human-like monkeys. General techniques for producing diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and Losman, M.J. et al, int.J. cancer 46:310, 1990. Serum containing immunologically active antibodies is then produced from the blood of such immunized animals using standard procedures well known in the art.

Monoclonal antibodies

In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody. anti-MASP-2 monoclonal antibodies are highly specific against a single MASP-2 epitope. As used herein, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring antibody production by any particular method. Monoclonal antibodies can be obtained using any technique that provides for antibody molecule production by a continuous cell line in culture, such as by the hybridoma method described by Kohler, g. Et al, nature 256:495, 1975, or they can be prepared by recombinant DNA methods (see, e.g., U.S. patent No. 4,816,567 to cabill). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in Clackson, T.et al, nature 352:624-628, 1991, and Marks, J.D. et al, J.mol. Biol.222:581-597, 1991. Such antibodies may have any immunoglobulin class, including IgG, igM, igE, igA, igD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., a BALB/C mouse) with a composition comprising a MASP-2 polypeptide or a portion thereof. After a predetermined period of time, spleen cells were removed from the mice and suspended in cell culture medium. The spleen cells are then fused with an immortalized cell line to form hybridomas. The hybridomas formed are grown in cell culture and screened for their ability to produce monoclonal antibodies directed against MASP-2. Examples are provided herein to further describe the generation of anti-MASP-2 monoclonal antibodies (see also Current Protocols in Immunology, volume 1, john Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies can be obtained by using transgenic mice that have been engineered to produce specific human antibodies in response to antigen challenge. In this technique, elements of human immunoglobulin heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line containing targeted disruption of endogenous immunoglobulin heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigen described herein, and the mice can be used to produce hybridomas that secrete human MASP-2 antibodies by fusing B cells from such animals with a suitable myeloma cell line using conventional Kohler-Milstein techniques, as described further herein. Transgenic mice having a human immunoglobulin genome are commercially available (e.g., from Abgenix, inc., fremont, CA and Medarex, inc., annandale, n.j.). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L. et al, nature Genet.7:13, 1994; lonberg, n. et al, nature 368:856, 1994; and Taylor, L.D. et al, int.Immun.6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such separation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography with protein A agarose (see, e.g., coligan on pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; baines et al, "Purification ofImmunoglobulin G (IgG)," at Methods in Molecular Biology, the Humana Press, inc., volume 10, pages 79-104, 1992).

Once generated, the polyclonal, monoclonal or phage-derived antibodies are first tested for specific MASP-2 binding. Antibodies that specifically bind to MASP-2 may be detected using a variety of assays known to those of skill in the art. Exemplary assays include Western blot or immunoprecipitation assays, immunoelectrophoresis, enzyme-linked immunosorbent assays, dot blots, inhibition or competition assays, and sandwich assays by standard methods (e.g., as described in Ausubel et al) (e.g., harlow and Land, antibodies: A Laboratory Manual, cold Spring Harbor Laboratory Press, 1988). Once antibodies that specifically bind to MASP-2 are identified, the ability of the anti-MASP-2 antibody to act as a MASP-2 inhibitor is tested in one of several assays, such as a lectin-specific C4 cleavage assay (described in example 2), a C3b deposition assay (described in example 2), or a C4b deposition assay (described in example 2).

The affinity of an anti-MASP-2 monoclonal antibody may be readily determined by one of ordinary skill in the art (see, e.g., scatchard, A., NY Acad. Sci.51:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies useful in the methods of the invention bind to MASP-2 with a binding affinity of <100nM, preferably <10nM and most preferably <2nM.

Chimeric/humanized antibodies

Monoclonal antibodies useful in the methods of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies (U.S. Pat. No. 4,816,567 to cabily; and Morrison, S.L. et al, proc.Nat' l Acad.Sci.USA 81:6851-6855, 1984).

One form of chimeric antibody useful in the present invention is a humanized monoclonal anti-MASP-2 antibody. Humanized versions of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Humanized monoclonal antibodies are generated by transferring non-human (e.g., mouse) Complementarity Determining Regions (CDRs) from the heavy and light chain variable regions of a mouse immunoglobulin into a human variable domain. Typically, the residues of the human antibody are then substituted in the framework regions of the non-human counterpart. In addition, the humanized antibody may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one variable domain, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Fv framework regions are of a human immunoglobulin sequence. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, P.T. et al, nature 321:522-525, 1986; reichmann, L. et al, nature 332:323-329, 1988; and Presta, curr.Op.struct.biol.2:593-596, 1992.

Humanized antibodies useful in the present invention include human monoclonal antibodies comprising at least the MASP-2 binding CDRH3 region. Alternatively, the Fc portion may be replaced so as to produce IgA or IgM as well as human IgG antibodies. Such humanized antibodies will have particular clinical utility because they will specifically recognize human MASP-2, but will not elicit an immune response in humans against the antibody itself. Thus, they are more suitable for in vivo administration in humans, especially when repeated administration or long-term administration is required.

Examples of the production of humanized anti-MASP-2 antibodies from murine anti-MASP-2 monoclonal antibodies are provided herein in example 6. Techniques for producing humanized monoclonal antibodies are also described, for example, by: jones, P.T. et al, nature 321:522, 1986; carter, P.et al, proc.Nat' l.Acad.Sci.USA 89:4285, 1992; sandhu, J.S., crit.Rev.Biotech.12:437, 1992; singer, I.I. et al, J.Immun.150:2844, 1993; sudhir (edit), antibody Engineering Protocols, humana Press, inc.,1995; kelley, "Engineering Therapeutic Antibodies," at Protein Engineering: principles and Practice, cleland et al (eds.), john Wiley & Sons, inc., pages 399-434, 1996; U.S. Pat. No. 5,693,762,1997 to Queen. In addition, there are commercial entities that synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain View, CA).

Recombinant antibodies

anti-MASP-2 antibodies may also be prepared using recombinant methods. For example, human antibodies can be prepared using a human immunoglobulin expression library (e.g., available from Stratagene, corp., la Jolla, CA) to produce fragments of human antibodies (VH, VL, fv, fd, fab or F (ab') 2). These fragments are then used to construct fully human antibodies using techniques similar to those used to generate chimeric antibodies.

Anti-idiotype antibody

Once anti-MASP-2 antibodies having the desired inhibitory activity are identified, these antibodies can be used to generate anti-idiotype antibodies that resemble a portion of MASP-2 using techniques well known in the art. See, for example, greenspan, N.S. et al, FASEB J.7:437, 1993. For example, antibodies that bind to MASP-2 and competitively inhibit the interaction of MASP-2 protein required for complement activation may be used to generate an anti-idiotype that resembles the MBL binding site on MASP-2 protein and thus bind to and neutralize the binding ligand of MASP-2, e.g., MBL.

Immunoglobulin fragments

MASP-2 inhibitors useful in the methods of the invention include not only intact immunoglobulin molecules, but also well-known fragments, including Fab, fab ', F (ab) 2, F (ab') 2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

It is well known in the art that only a small portion of antibody molecules, para (paratope), are involved in binding of antibodies to their epitopes (see e.g., clark, w.r., the Experimental Foundations of Modern Immunology, wiley & Sons, inc., NY, 1986). The pFc' and Fc regions of antibodies are effectors of the classical complement pathway, but are not involved in antigen binding. Antibodies from which the pFC ' region has been enzymatically cleaved, or which have produced no pFC ' region, are designated as F (ab ') 2 fragments, and retain the two antigen binding sites of the intact antibody. The isolated F (ab') 2 fragment is referred to as a bivalent monoclonal fragment due to its two antigen binding sites. Similarly, antibodies from which the Fc region has been enzymatically cleaved, or which have been raised to be free of the Fc region, are designated as Fab fragments, and retain one of the antigen binding sites of the intact antibody molecule.

Antibody fragments may be obtained by proteolytic hydrolysis, for example by pepsin or papain digestion of the whole antibody by conventional methods. For example, antibody fragments may be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F (ab') 2. Such fragments may be further cleaved using thiol reducing agents to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction may be performed using a protecting group derived from disulfide-bonded cleaved sulfhydryl groups. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in the following: U.S. Pat. No. 4,331,647 to Goldenberg; nisonoff, A. Et al, arch. Biochem. Biophys.89:230, 1960; porter, R.R., biochem.J.73:119, 1959; edelman et al, methods in Enzymology 1:422,Academic Press,1967; and pages 2.8.1-2.8.10 and 2.10.-2.10.4 by Coligan.

In some embodiments, it is preferred to use antibody fragments lacking an Fc region to avoid activation of the classical complement pathway that is initiated upon binding of Fc to fcγ receptors. There are several methods by which moabs can be generated that avoid fcγ receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed using partial digestion by proteolytic enzymes (e.g., ficin digestion) to generate, for example, antigen-binding antibody fragments, such as Fab or F (ab) 2 fragments (Mariani, M. Et al, mol. Immunol.28:69-71, 1991). Alternatively, human gamma 4IgG isotypes that do not bind fcgamma receptor may be used during the construction of humanized antibodies as described herein. Antibodies, single chain antibodies, and antigen binding domains lacking an Fc domain can also be engineered using recombinant techniques described herein.

Single chain antibody fragments

Alternatively, a single peptide chain binding molecule specific for MASP-2 may be produced in which the heavy and light chain Fv regions are linked. Fv fragments may be joined by a peptide linker to form a single chain antigen binding protein (scFv). These single chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding VH and VL domains linked by oligonucleotides. The structural gene is inserted into an expression vector which is then introduced into a host cell, such as E.coli (E.coli). Recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for generating scFv are described, for example, by: whitlow et al, "Methods: A Companion to Methods in Enzymology"2:97, 1991; bird et al, science 242:423, 1988; U.S. Pat. nos. 4,946,778 to Ladner; pack, P. Et al, bio/Technology 11:1271, 1993.

As an illustrative example, MASP-2 specific scFv may be obtained by exposing lymphocytes to MASP-2 polypeptides in vitro, and selecting an antibody display library in a phage or similar vector (e.g., by using immobilized or labeled MASP-2 proteins or peptides). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains may be obtained by screening random peptide libraries displayed on phage or bacteria (e.g., E.coli). These random peptide display libraries can be used to screen peptides that interact with MASP-2. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. Pat. No. 5,223,409 to Lardner; U.S. Pat. No. 4,946,778 to Lardner; U.S. Pat. No. 5,403,484 to Lardner; U.S. Pat. No. 5,571,698 to Lardner; and Kay et al Phage Display of Peptides and Proteins Academic Press, inc., 1996), and random peptide display libraries and kits for screening such libraries are commercially available, for example, from CLONTECH Laboratories, inc. (Palo Alto, calif.), invitrogen Inc. (San Diego, calif.), new England Biolabs, inc. (Ipswich, mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

Another form of anti-MASP-2 antibody fragment that may be used in this aspect of the invention is a peptide encoding a single Complementarity Determining Region (CDR) that binds to an epitope on the MASP-2 antigen and inhibits MASP-2 dependent complement activation. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding CDRs of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction from the RNA synthesis variable region of antibody-producing cells (see, e.g., larrick et al, methods: A Companion to Methods in Enzymology: 106, 1991; courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," at Monoclonal Antibodies: production, engineering and Clinical Application, ritter et al (eds.), page 166, cambridge University Press,1995; and Ward et al, "Genetic Manipulation and Expression of Antibodies," at Monoclonal Antibodies: principles and Applications, birch et al (eds.), page 137, wiley-Lists, inc., 1995).

The MASP-2 antibodies described herein are administered to a subject in need thereof to inhibit MASP-2 dependent complement activation. In some embodiments, the MASP-2 inhibitor is a high affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

Peptide inhibitors

In some embodiments of this aspect of the invention, the MASP-2 inhibitor comprises an isolated MASP-2 peptide inhibitor, including isolated natural peptide inhibitors and synthetic peptide inhibitors, which inhibit the MASP-2 dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" refers to a peptide that inhibits MASP-2 dependent complement activation by: binding to MASP-2, competing with MASP-2 for binding to another recognition molecule in the lectin pathway (e.g., MBL, H-fiber gel protein, M-fiber gel protein, or L-fiber gel protein), and/or interacting directly with MASP-2 to inhibit MASP-2-dependent complement activation, the peptides are substantially pure and substantially free of other substances with which they may be found in nature to the extent practical and appropriate for their intended use.

Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors directed against adhesion molecules structurally related to LFA 1 have recently been approved for clinical use in coagulopathies (ohm an, E.M. et al, european Heart J.16:50-55, 1995). Short linear peptides (< 30 amino acids) that prevent or interfere with integrin-dependent adhesion have been described (Murayama, O.et al J.biochem.120:445-51, 1996). Longer peptides ranging in length from 25 to 200 amino acid residues have also been successfully used to block integrin-dependent adhesion (Zhang, l. Et al, j. Biol. Chem.271 (47): 29953-57, 1996). In general, longer peptide inhibitors have higher affinity and/or slower dissociation rates than short peptides, and thus may be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be potent inhibitors of integrins in vivo for the treatment of human inflammatory diseases (Jackson, D.Y. et al, J.Med. Chem.40:3359-68, 1997). One method of producing cyclic peptides involves the synthesis of peptides in which the terminal amino acids of the peptide are cysteines, allowing the peptide to exist in cyclic form through disulfide bonding between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasms (e.g., U.S. patent No. 6,649,592 to Larson).

Synthesis of MASP-2 peptide inhibitors

MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic target regions important for MASP-2 function. Inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides that may be used in the practice of this aspect of the invention. Candidate MASP-2 inhibitory peptides may be tested for their ability to act as MASP-2 inhibitors in one of several assays, including, for example, lectin-specific C4 cleavage assays (described in example 2) and C3b deposition assays (described in example 2).

In some embodiments, the MASP-2 inhibitory peptide is derived from a MASP-2 polypeptide and is selected from the group consisting of the full length mature MASP-2 protein (SEQ ID NO: 6), or a specific domain of the MASP-2 protein, such as the CUBI domain (SEQ ID NO: 8), the CUBIEGF domain (SEQ ID NO: 9), the EGF domain (SEQ ID NO: 11), and the serine protease domain (SEQ ID NO: 12). As previously described, the CUBEGFCUBII region has been shown to be necessary for dimerization and binding to MBL (Thielens et al, supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified persons carrying homozygous mutations at Asp105 to Gly105, resulting in loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K. Et al, new England J.Med.349:554-560, 2003).

In some embodiments, the MASP-2 inhibitory peptide is derived from a lectin protein that binds to MASP-2 and is involved in the lectin complement pathway. Several different lectins involved in this pathway have been identified, including mannan-binding lectin (MBL), L-fiber gelator, M-fiber gelator, and H-fiber gelator. (Ikeda, K. Et al, J.biol. Chem.262:7451-7454, 1987; matsushita, M. Et al, J.exp. Med.176:1497-2284, 2000; matsushita, M. Et al, J.Immunol.168:3502-3506, 2002). These lectins are present in serum as oligomers of homotrimeric subunits, each having N-terminal collagen-like fibers containing a carbohydrate recognition domain. These different lectins have been shown to bind to MASP-2 and the lectin/MASP-2 complex activates complement by cleavage of proteins C4 and C2. H-fiber gelator has an amino terminal region of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M. Et al, J. Immunol.168:3502-3506, 2002). H-fiber gellin binds to GlcNAc and aggregates human erythrocytes coated with LPS derived from salmonella typhimurium (s.tyrphimum), salmonella minnesota (s.minnesota) and escherichia coli. H-fiber gelator has been shown to bind MASP-2 and MAP19 and activate the lectin pathway. As above. L-fiber gelator/P35 also binds to GlcNAc and has been shown to bind to MASP-2 and MAp19 in human serum and the complex has been shown to activate the lectin pathway (Matsushita, M. Et al, J. Immunol.164:2281, 2000). Thus, MASP-2 inhibitory peptides useful in the present invention may comprise a region of at least 5 amino acids selected from the group consisting of: MBL protein (SEQ ID NO: 21), H-fiber gelator (Genbank accession NM-173452), M-fiber gelator (Genbank accession O00602) and L-fiber gelator (Genbank accession NM-015838).

More specifically, scientists have identified MASP-2 binding sites on MBL within the 12 Gly-X-Y triplets "GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POG NOGPSG SOG PKG QKG DOG KS" (SEQ ID NO: 26) that are located between the hinge and neck of the C-terminal portion of the collagen-like domain of MBP (Wallis, R. Et al, J.biol. Chem.279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-fiber gel protein and human L-fiber gel protein. Consensus binding sites have been described which are present in all three lectin proteins comprising the amino acid sequence "OGK-X-GP" (SEQ ID NO: 22), wherein the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al, 2004, supra). Thus, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO. 22. Peptides derived from MBL comprising the amino acid sequence "GLR GLQ GPO GKL GPO G" (SEQ ID NO: 24) have been shown to bind MASP-2 in vitro (Wallis et al, 2004, supra). To enhance binding to MASP-2, peptides flanked by two GPO triplets at each end may be synthesized ("GPO GPO GLR GLQ GPO GKL GPO GGP OGP O" SEQ ID NO: 25) to enhance triple helix formation as found in native MBL proteins (as further described in Wallis, R. Et al, J. Biol. Chem.279:14065, 2004).

MASP-2 inhibitory peptides may also be derived from human H-fiber gelator protein, which includes the sequence "GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO" (SEQ ID NO: 27) from the consensus MASP-2 binding region in H-fiber gelator protein. Also included are peptides derived from human L-fiber gel protein, which include the sequence "GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO" (SEQ ID NO: 28) from the consensus MASP-2 binding region in L-fiber gel protein.

MASP-2 inhibitory peptides may also be derived from a C4 cleavage site, such as "LQRALEILPNRVTIKANRPFLVFI" (SEQ ID NO: 29), which is a C4 cleavage site linked to the C-terminal portion of antithrombin III (Glover, G.I. et al, mol.Immunol.25:1261 (1988)).

Table 3: exemplary MASP-2 inhibitory peptides

Note that: the letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

Peptides derived from the C4 cleavage site, as well as other peptides that inhibit the MASP-2 serine protease site, may be chemically modified so that they are irreversible protease inhibitors. For example, suitable modifications may include, but are not necessarily limited to, halomethyl ketone (Br, cl, I, F) at the C-terminus, asp, or Glu, or attached to a functional side chain; haloacetyl (or other alpha-haloacetyl) groups on amino or other functional side chains; epoxide-or imine-containing groups at the amino or carboxyl terminus or on functional side chains; or imidoesters at the amino or carboxyl terminus or on functional side chains. Such modifications provide the advantage of permanently inhibiting the enzyme by covalent attachment of the peptide. This may result in lower effective doses and/or less frequent administration of the peptide inhibitor is required.

In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the methods of the invention include peptides comprising MASP-2 binding CDRH3 regions of anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR regions for the synthetic peptides can be determined by methods known in the art. The heavy chain variable region is a peptide typically ranging from 100 to 150 amino acids in length. The light chain variable region is a peptide typically ranging from 80 to 130 amino acids in length. CDR sequences within the heavy and light chain variable regions comprise only about 3-25 amino acid sequences, which can be readily sequenced by one of ordinary skill in the art.

One of skill in the art will recognize that substantially homologous variations of the above MASP-2 inhibitory peptides also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, deletions, substitutions, and/or additional amino acids at the carboxy-terminal or amino-terminal portion of the subject peptide, and mixtures thereof. Thus, those homologous peptides having MASP-2 inhibitory activity are considered useful in the methods of the invention. The peptides may also include repeated motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein and include the exchange of an amino acid for another amino acid of similar charge, size, or hydrophobicity, etc.

MASP-2 inhibitory peptides may be modified to increase solubility and/or to maximize positive or negative charges to more closely resemble segments in an intact protein. The derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, provided that the derivatives functionally retain the desired properties of MASP-2 inhibition. Modifications may include amino acid substitutions with one of the twenty commonly known amino acids or another amino acid, amino acid substitutions with a derivative or substituted amino acid having an auxiliary desired characteristic (e.g., resistance to enzymatic degradation), or amino acid substitutions with a D amino acid, or substitutions with another molecule or compound, e.g., a carbohydrate, that mimics the native conformation and function of one or more amino acids or peptides; amino acid deletion; amino acid insertions with one or the other of the twenty commonly known amino acids, amino acid insertions with derivatisation or insertion amino acids that assist the desired characteristics (e.g. resistance to enzymatic degradation), or amino acid insertions with D amino acids, or substitutions with another molecule or compound, e.g. a carbohydrate, which mimics the native conformation and function of one or more amino acids or peptides; or substitution with another molecule or compound, such as a carbohydrate or nucleic acid monomer, that mimics the native conformation, charge distribution and function of the parent peptide. Peptides may also be modified by acetylation or amidation.

The synthesis of the derivative inhibitory peptide may depend on known techniques of peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, the skilled person may rely on a suitable computer program to determine the conformation of the peptide of interest. Once the conformation of the peptides disclosed herein is known, the skilled artisan can determine in a rational design manner which substitutions can be made at one or more sites to prepare derivatives that retain the basic conformation and charge distribution of the parent peptide, but which may have characteristics that are not present in or enhanced beyond those found in the parent peptide. Once candidate derivative molecules are identified, the derivatives can be tested using the assays described herein to determine if they act as MASP-2 inhibitors.

Screening for MASP-2 inhibitory peptides

Peptides that mimic the molecular structure of the critical binding regions of MASP-2 and inhibit complement activity of MASP-2 can also be generated and screened using molecular modeling and rational molecular design. The molecular structure used for modeling includes the CDR regions of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including the regions required for dimerization, the regions involved in binding to MBL, and serine protease active sites, as previously described. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting can be used to construct macromolecular structures from the head, such as peptides that bind to specific molecules. See, e.g., shea, k.j., "Molecular Imprinting of Synthetic Network Polymers: the De Novo synthesis of Macromolecular Binding and Catalytic Sties," TRIP 2 (5) 1994.

As an illustrative example, one method of preparing a mimetic of MASP-2 binding peptide is as follows. Functional monomers of known MASP-2 binding peptides or the binding region (template) of anti-MASP-2 antibodies exhibiting MASP-2 inhibition are polymerized. The template is then removed, followed by polymerization of the second type of monomer in the void left by the template, to provide a new molecule that exhibits one or more desired properties similar to the template. In addition to preparing peptides in this manner, other MASP-2 binding molecules may be prepared that are inhibitors of MASP-2, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other bioactive materials. Such methods can be used to design a wide variety of biomimetics that are more stable than their natural counterparts, as they are typically prepared by free radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

Peptide synthesis

MASP-2 inhibitory peptides may be prepared using techniques well known in the art, such as the solid phase synthesis technique described initially by Merrifield in J.Amer.chem.Soc.85:2149-2154, 1963. Automated synthesis may be accomplished according to instructions provided by the manufacturer, for example using Applied Biosystems 431A Peptide Synthesizer (Foster City, calif.). Other techniques may be found, for example, in Bodanszky, M.et al, peptide Synthesis, second edition, john Wiley & Sons,1976, and other references known to those skilled in the art.

Peptides may also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, peptides may be enzymatically produced by: inserting a nucleic acid encoding a peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the desired amino acid. The peptide is then purified using chromatographic or electrophoretic techniques or by means of a carrier protein which can be fused to and subsequently cleaved from the peptide by inserting the nucleic acid sequence encoding the carrier protein into the expression vector in phase with the peptide coding sequence. The fusion protein-peptide may be isolated using chromatography, electrophoresis, or immunological techniques (e.g., via antibody to carrier protein binding to a resin). The peptides may be cleaved using chemical methods or as by enzymatic cleavage, e.g. by hydrolytic enzymes.

MASP-2 inhibitory peptides useful in the methods of the invention may also be produced in recombinant host cells following conventional techniques. In order to express the MASP-2 inhibitory peptide coding sequence, the nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences such as promoters and enhancers, expression vectors may include translational regulatory sequences and marker genes suitable for selection of cells carrying the expression vector.

Nucleic acid molecules encoding MASP-2 inhibitory peptides may be synthesized using protocols such as the phosphoramidite method using "genetic machinery". If chemically synthesized double-stranded DNA is required for synthesis using, for example, a gene or a gene fragment, each complementary strand is prepared separately. The generation of short genes (60 to 80 base pairs) is technically simple and can be accomplished by synthesizing the complementary strand and then annealing it. For the production of longer genes, the synthetic genes (double strand) are assembled in modular form from single strand fragments, which are 20 to 100 nucleotides in length. For reviews of polynucleotide synthesis, see, e.g., glick and Pasternak, "Molecular Biotechnology, principles and Applications of Recombinant DNA", ASM Press,1994; itakura, K. Et al, annu. Rev. Biochem.53:323, 1984; and Climie, S.et al, proc.Nat' l Acad.Sci.USA 87:633, 1990.

Small molecule inhibitors of MASP-2

In some embodiments, MASP-2 inhibitors are small molecule inhibitors, including natural, semisynthetic and synthetic materials having low molecular weights (e.g., 50 to 1000 Da), such as peptides, peptidomimetics and non-peptide inhibitors (e.g., oligonucleotides and organic compounds). Small molecule inhibitors of MASP-2 may be generated based on the molecular structure of the variable region of the anti-MASP-2 antibody.

The use of computational drug design can also be used to design and generate small molecule inhibitors based on the crystal structure of MASP-2 (Kuntz I.D. et al, science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H. Et al, EMBO J.22:2348-2359, 2003). Using the method described by Kuntz et al, MASP-2 crystal structure coordinates are used as input to a computer program such as DOCK, which outputs a list of small molecule structures that are expected to bind to MASP-2. The use of such computer programs is well known to those skilled in the art. For example, by using the procedure DOCK (Kuntz, I.D. et al, J.mol. Biol.161:269-288, 1982; desJarlais, R.L. et al, PNAS 87:6644-6648, 1990), the fit of compounds found in the Cambridge Crystallographic database to enzyme binding sites was evaluated for the crystal structure of HIV-1 protease inhibitors to identify unique non-peptide ligands that are HIV-1 protease inhibitors.

Exemplary MASP-2 inhibitors include, but are not limited to, the compounds disclosed in U.S. patent application Ser. Nos. 62/943,629, 62/943,622, 62/943,611, 62/943,599, 16/425,791 and PCT application Ser. No. PCT/US19/34220, each of which is incorporated herein by reference in its entirety.

In some embodiments, the small molecule is a compound of formula (I-1), (IIA), (IIB), (III), or (IV):

or a salt thereof, wherein:

Cy ^1A is unsubstituted or substituted C _6-10 Aryl, or unsubstituted or substituted 5-10 membered heteroaryl; in which Cy is formed ^1A The ring atoms of the 5-10 membered heteroaryl group consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; in which Cy is formed ^1A Substituted C of (2) _6-10 Aryl or substituted 5-10 membered heteroaryl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy1A Halogen, C _1-6 Haloalkyl, CN, OR ^a11 、S ^Ra11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、C(＝NOR ^a11 )NR ^c11 R ^d11 、C(＝NOC(O)R ^b11 )NR ^c11 R ^d11 、C(＝NR ^e11 )NR ^c11 C(O)OR ^a11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

each R ^Cy1A Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy1A The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R is formed ^Cy1A Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo, and wherein R is formed ^Cy1A Each C of (2) _6-10 Aryl group5-to 10-membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

R ¹¹ is H or C _1-6 Alkyl, C _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl, wherein R is formed ¹¹ C of (2) _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo, and wherein R is formed ¹¹ C of (2) _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

R ¹² is H or C _1-6 An alkyl group; or (b)

R ¹¹ And R is ¹² Together with the groups to which they are attached form a 4-6 membered heterocycloalkyl ring;

A ¹¹ is CR (CR) ¹³ R ¹⁵ Or N;

each R ¹³ Independently Cy ^1B 、(CR ^13A R ^13B ) _n3 Cy ^1B 、(C _1-6 Alkylene) Cy ^1B 、(C _2-6 Alkenylene) Cy ^1B 、(C _2-6 Alkynylene) Cy ^1B Or OCy ^1B Wherein R is ¹³ C of (2) _1-6 Alkylene, C _2-6 Alkenylene or C _2-6 The alkynylene component is unsubstituted OR substituted with 1, 2, 3, 4 OR 5 substituents each independently selected from halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

each R ¹⁴ Independently selected from H and C _1-6 An alkyl group;

R ¹⁵ selected from H, R ¹³ 、C _1-6 Alkyl and OH;

a pair of R's attached to adjacent carbon atoms ¹⁴ A group, or a pair of R's attached to adjacent carbon atoms ¹⁴ And R is ¹⁵ The radicals being independently of R ¹⁴ Together are replaced by connecting the pair of R ¹⁴ A group or the pair of R ¹⁴ And R is ¹⁵ A bond to an adjacent carbon atom to which the group is attached such that the adjacent carbon atom is attached by a double bond; or (b)

A pair of R's attached to the same carbon atom ¹⁴ A group, or a pair of R's bound to the same carbon atom ¹³ And R is ¹⁵ The radicals being independently of R ¹⁴ And together with the pair of R ¹⁴ A group or the pair of R ¹³ And R is ¹⁵ The radicals together with the carbon atoms to which they are attached forming a spiro-fused C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the 4-10 membered heterocycloalkyl ring formed consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein the spiro-condensed C formed _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo; or (b)

A pair of R's attached to adjacent carbon atoms ¹⁴ A group, or a pair of R's attached to adjacent carbon atoms ¹⁴ And R is ¹⁵ The radicals being independently of R ¹⁴ Along with other occurrences of the pair of R ¹⁴ A group or the pair of R ¹⁴ And R is ¹⁵ The radicals together with the adjacent carbon atoms to which they are attached forming a condensed C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the 4-10 membered heterocycloalkyl ring formed consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein the fused C is formed _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo; or (b)

A group of four R's linked to two adjacent carbon atoms ¹⁴ A group, or a group of two R's attached to two adjacent carbon atoms ¹⁴ One of (a)R ¹³ And one R ¹⁵ The radicals being independently of R ¹⁴ Along with the other occurrences of the set of four R ¹⁴ A group, or the group of two R ¹⁴ One R ¹³ And one R ¹⁵ The groups being taken together with the two adjacent carbon atoms to which they are attached to form a fused C _6-10 Aryl or 5-to 10-membered heteroaryl, C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, wherein the ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl ring formed consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, and wherein the fused C formed _6-10 Aryl or 5-to 10-membered heteroaryl, C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl ring, optionally further substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2- 6 alkenyl, C _2-6 Alkynyl, haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

n1 is 1 or 2;

n2 is 0, 1 or 2;

provided that the sum of n1 and n2 is 1, 2 or 3;

provided that if n1 is 1 or n2 is 0, A ¹¹ Is CR (CR) ¹³ R ¹⁵ ；

n3 is 0, 1 or 2;

each R ^13A Independently H or C _1-6 An alkyl group;

each R ^13B Independently H or C _1-6 An alkyl group; or (b)

Or R attached to the same carbon atom ^13A And R is ^13B Independent of any other R ^13A And R is ^13B Groups taken together may form- (CH) ₂ ) _2-5 -, thereby forming a 3-6 membered cycloalkyl ring; or (b)

Cy ^1B Is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^1B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

in which Cy is formed ^1B Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy1B Halogen, C _1-6 Haloalkyl, CN, OR ^a11 、S ^Ra11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、C(＝NOR ^a11 )NR ^c11 R ^d11 、C(＝NOC(O)R ^b11 )NR ^c11 R ^d11 、C(＝NR ^e11 )NR ^c11 C(O)OR ^a11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

wherein each R is ^Cy1B Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy1B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R is formed ^Cy1B Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O) ^Rb11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo; and wherein R is formed ^Cy1B Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O) ^Rb11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

R ¹⁶ h, cy of a shape of H, cy ^1C 、C _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl, wherein R is formed ¹⁶ C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: cy (Cy) ^1C Halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo, provided that R ¹⁶ Not more than one substituent of (C) is Cy ^1C ；

Cy ^1C Is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^1C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

in which Cy is formed ^1C Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy1C Halogen, C _1-6 Haloalkyl, CN, OR ^a11 、S ^Ra11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O)R ^b11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、C(＝NOR ^a11 )NR ^c11 R ^d11 、C(＝NOC(O)R ^b11 )NR ^c11 R ^d11 、C(＝NR ^e11 )NR ^c11 C(O)OR ^a11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

wherein each R is ^Cy1C Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy1C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R is formed ^Cy1C Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O) ^Rb11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo; and wherein R is formed ^Cy1C Each C of (2) _6-10 Aryl, 5-10 memberedHeteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a11 、SR ^a11 、C(O)R ^b11 、C(O)NR ^c11 R ^d11 、C(O)OR ^a11 、OC(O)R ^b11 、OC(O)NR ^c11 R ^d11 、NR ^c11 R ^d11 、NR ^c11 C(O) ^Rb11 、NR ^c11 C(O)NR ^c11 R ^d11 、NR ^c11 C(O)OR ^a11 、C(＝NR ^e11 )NR ^c11 R ^d11 、NR ^c11 C(＝NR ^e11 )NR ^c11 R ^d11 、S(O)R ^b11 、S(O)NR ^c11 R ^d11 、S(O) ₂ R ^b11 、NR ^c11 S(O) ₂ R ^b11 、S(O) ₂ NR ^c11 R ^d11 And oxo;

R ^a11 、R ^b11 、R ^c11 and R is ^d11 Each independently selected from H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a11 、R ^b11 、R ^c11 And R is ^d11 Is not less than C _1- 6 alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, halogen, CN, OR ^a12 、SR ^a12 、C(O)R ^b12 、C(O)NR ^c12 R ^d12 、C(O)OR ^a12 、OC(O)R ^b12 、OC(O)NR ^c12 R ^d12 、NR ^c12 R ^d12 、NR ^c12 C(O)R ^b12 、NR ^c12 C(O)NR ^c12 R ^d12 、NR ^c12 C(O)OR ^a12 、C(＝NR ^e12 )NR ^c12 R ^d12 、NR ^c12 C(＝NR ^e12 )NR ^c12 R ^d12 、S(O)R ^b12 、S(O)NR ^c12 R ^d12 、S(O) ₂ R ^b12 、NR ^c12 S(O) ₂ R ^b12 、S(O) ₂ NR ^c12 R ^d12 And oxo;

or R attached to the same N atom ^c11 And R is ^d11 Together with the N atom to which they are both attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: c (C) _1-6 Alkyl, halogen, CN, OR ^a12 、SR ^a12 、C(O)R ^b12 、C(O)NR ^c12 R ^d12 、C(O)OR ^a12 、OC(O)R ^b12 、OC(O)NR ^c12 R ^d12 、NR ^c12 R ^d12 、NR ^c12 C(O)R ^b12 、NR ^c12 C(O)NR ^c12 R ^d12 、NR ^c12 C(O)OR ^a12 、C(＝NR ^e12 )NR ^c12 R ^d12 、NR ^c12 C(＝NR ^e12 )NR ^c12 R ^d12 、S(O)R ^b12 、S(O)NR ^c12 R ^d12 、S(O) ₂ R ^b12 、NR ^c12 S(O) ₂ R ^b12 、S(O) ₂ NR ^c12 R ^d12 And oxo;

R ^a12 、R ^b12 、R ^c12 and R is ^d12 Each independently selected from H, C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a12 、R ^b12 、R ^c12 And R is ^d12 Is not less than C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

or R attached to the same N atom ^c12 And R is ^d12 Together with the N atom to which they are each attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each of which is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

R ^e11 and Re (Re) ¹² Each independently is H, CN or NO ₂ ；

Cy ^2A Is unsubstituted or substituted C _6-10 Aryl, or unsubstituted or substituted 5-10 membered heteroaryl; in which Cy is formed ^2A The ring atoms of the 5-10 membered heteroaryl group consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; in which Cy is formed ^2A Substituted C of (2) _6-10 Aryl or substituted 5-10 membered heteroaryl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy2A Halogen, C _1-6 Haloalkyl, CN, OR ^a21 、S ^Ra21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、C(＝NOR ^a21 )NR ^c21 R ^d21 、C(＝NOC(O)R ^b21 )NR ^c21 R ^d21 、C(＝NR ^e21 )NR ^c21 C(O)OR ^a21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

each R ^Cy2A Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy2A The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R is formed ^Cy2A Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo, wherein R is formed ^Cy2A Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

R ²¹ is H or C _1-6 Alkyl, C _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl, wherein R is formed ²¹ C of (2) _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo, and wherein R is formed ²¹ C of (2) _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl radicals being unsubstitutedOr substituted with 1, 2 or 3 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

R ²² is H or C _1-6 An alkyl group; or (b)

R ²¹ And R is ²² Together with the groups to which they are attached form a 4-6 membered heterocycloalkyl ring;

A ²³ is N or NR ²³ ；

A ²⁴ Is CR (CR) ²⁴ N and NR ²⁴ ；

A ²⁶ Is CR (CR) ²⁶ Or S;

provided that it is

A in formula (IIA) ²³ 、A ²⁴ And A ²⁶ Selected so as to contain A ²³ 、A ²⁴ And A ²⁶ Is a heteroaryl ring, and is signed withRepresents an aromatic ring (standardized) bond;

R ²³ is H or C _1-6 An alkyl group;

R ²⁴ is H; c (C) _1-6 Alkyl or phenyl;

R ²⁵ is Cy ^2B 、(CR ^25A R ^25B ) _n25 Cy ^2B 、(C _1-6 Alkylene) Cy ^2B 、(C _2-6 Alkenylene) Cy ^2B Or (C) _2-6 Alkynylene) Cy ^2B Wherein R is ²⁵ C of (2) _1-6 Alkylene, C _2-6 Alkenylene or C _2-6 The alkynylene component is unsubstituted OR substituted with 1, 2, 3, 4 OR 5 substituents each independently selected from halogen, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

R ²⁶ is H or C _1-6 An alkyl group;

each R ^25A Is H or C _1-6 An alkyl group;

each R ^25B Is H or C _1-6 An alkyl group;

n25 is 0, 1 or 2;

Cy ^2B is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^2B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

in which Cy is formed ^2B Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy2B Halogen, C _1-6 HaloalkanesRadical, CN, OR ^a21 、S ^Ra21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O)R ^b21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、C(＝NOR ^a21 )NR ^c21 R ^d21 、C(＝NOC(O)R ^b21 )NR ^c21 R ^d21 、C(＝NR ^e21 )NR ^c21 C(O)OR ^a21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

wherein each R is ^Cy2B Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy2B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R is formed ^Cy2B Each C of (2) _1-6 Alkyl, C _2- 6 alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O) ^Rb21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo; and wherein R is formed ^Cy2B Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a21 、SR ^a21 、C(O)R ^b21 、C(O)NR ^c21 R ^d21 、C(O)OR ^a21 、OC(O)R ^b21 、OC(O)NR ^c21 R ^d21 、NR ^c21 R ^d21 、NR ^c21 C(O) ^Rb21 、NR ^c21 C(O)NR ^c21 R ^d21 、NR ^c21 C(O)OR ^a21 、C(＝NR ^e21 )NR ^c21 R ^d21 、NR ^c21 C(＝NR ^e21 )NR ^c21 R ^d21 、S(O)R ^b21 、S(O)NR ^c21 R ^d21 、S(O) ₂ R ^b21 、NR ^c21 S(O) ₂ R ^b21 、S(O) ₂ NR ^c21 R ^d21 And oxo;

each independently selected from H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a21 、R ^b21 、R ^c21 And R is ^d21 Is not less than C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, halogen, CN, OR ^a22 、SR ^a22 、C(O)R ^b22 、C(O)NR ^c22 R ^d22 、C(O)OR ^a22 、OC(O)R ^b22 、OC(O)NR ^c22 R ^d22 、NR ^c22 R ^d22 、NR ^c22 C(O)R ^b22 、NR ^c22 C(O)NR ^c22 R ^d22 、NR ^c22 C(O)OR ^a22 、C(＝NR ^e22 )NR ^c22 R ^d22 、NR ^c22 C(＝NR ^e22 )NR ^c22 R ^d22 、S(O)R ^b22 、S(O)NR ^c22 R ^d22 、S(O) ₂ R ^b22 、NR ^c22 S(O) ₂ R ^b22 、S(O) ₂ NR ^c22 R ^d22 And oxo;

or R attached to the same N atom ^c21 And R is ^d21 Together with the N atom to which they are both attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: c (C) _1-6 Alkyl, halogen, CN, OR ^a22 、SR ^a22 、C(O)R ^b22 、C(O)NR ^c22 R ^d22 、C(O)OR ^a22 、OC(O)R ^b22 、OC(O)NR ^c22 R ^d22 、NR ^c22 R ^d22 、NR ^c22 C(O)R ^b22 、NR ^c22 C(O)NR ^c22 R ^d22 、NR ^c22 C(O)OR ^a22 、C(＝NR ^e22 )NR ^c22 R ^d22 、NR ^c22 C(＝NR ^e22 )NR ^c22 R ^d22 、S(O)R ^b22 、S(O)NR ^c22 R ^d22 、S(O) ₂ R ^b22 、NR ^c22 S(O) ₂ R ^b22 、S(O) ₂ NR ^c22 R ^d22 And oxo;

R ^a22 、R ^b22 、R ^c22 and R is ^d22 Each independently selected from H, C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5 -6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a22 、R ^b22 、R ^c22 And R is ^d22 Is not less than C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

or R attached to the same N atom ^c22 And R is ^d22 Together with the N atom to which they are each attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each of which is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

R ^e21 and Re (Re) ²² Each independently is H, CN or NO ₂ ；

Cy ^3A Is unsubstituted or substituted C _6-10 Aryl, or unsubstituted or substituted 5-10 membered heteroaryl; in which Cy is formed ^3A The ring atoms of the 5-10 membered heteroaryl group consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; in which Cy is formed ^3A Substituted C of (2) _6-10 Aryl or substituted 5-10 membered heteroaryl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy3A Halogen, C _1-6 Haloalkyl, CN, OR ^a31 、S ^Ra31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、C(＝NOR ^a31 )NR ^c31 R ^d31 、C(＝NOC(O)R ^b31 )NR ^c31 R ^d31 、C(＝NR ^e31 )NR ^c31 C(O)OR ^a31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

each R ^Cy3A Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy3A The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R is formed ^Cy3A Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo, and wherein R is formed ^Cy3A Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

R ³¹ is H or C _1-6 Alkyl, C _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl, wherein R is formed ³¹ C of (2) _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo, and wherein is formedR ³¹ C of (2) _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

R ³² is H or C _1-6 An alkyl group; or (b)

R ³¹ And R is ³² Together with the groups to which they are attached form a 4-6 membered heterocycloalkyl ring;

R ³³ is Cy ^3B 、(CR ^33A R ^33B ) _n33 Cy ^3B 、(C _1-6 Alkylene) Cy ^3B 、(C _2-6 Alkenylene) Cy ^3B Or (C) _2-6 Alkynylene) Cy ^3B Wherein R is ³⁵ C of (2) _1-6 Alkylene, C _2-6 Alkenylene or C _2-6 The alkynylene component is unsubstituted OR substituted with 1, 2, 3, 4 OR 5 substituents each independently selected from halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

each R ^33A Independently H or C _1-6 An alkyl group;

each R ^33B Independently H or C _1-6 An alkyl group; or (b)

Or R attached to the same carbon atom ^33A And R is ^33B Independent of any other R ^33A And R is ^33B Groups taken together may form- (CH) ₂ ) _2-5 -, thereby forming a 3-6 membered cycloalkyl ring; or (b)

n33 is 0, 1, 2 or 3;

Cy ^3B is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^3B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; and

In which Cy is formed ^3B Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy3B Halogen, C _1-6 Haloalkyl, CN, OR ^a31 、S ^Ra31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、C(＝NOR ^a31 )NR ^c31 R ^d31 、C(＝NOC(O)R ^b31 )NR ^c31 R ^d31 、C(＝NR ^e31 )NR ^c31 C(O)OR ^a31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

wherein each R is ^Cy3B Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy3B The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R is formed ^Cy3B Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O) ^Rb31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo; and wherein R is formed ^Cy3B Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O) ^Rb31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

R ³⁴ selected from H and C _1-6 An alkyl group;

R ³⁵ selected from H, substituted or substituted C _1-6 Alkyl and Cy ^3C Wherein R is formed ³⁵ Substituted C of (2) _1-6 Alkyl is substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: cy (Cy) ^3C Halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo; provided that R ³⁵ Not more than one substituent of (C) is Cy ^3C ；

Cy ^3C Is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^3C The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl groups being composed of carbon atoms1, 2 or 3 heteroatoms selected from O, N and S; and

in which Cy is formed ^3C Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or substituted 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy3C Halogen, C _1-6 Haloalkyl, CN, OR ^a31 、S ^Ra31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O)R ^b31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、C(＝NOR ^a31 )NR ^c31 R ^d31 、C(＝NOC(O)R ^b31 )NR ^c31 R ^d31 、C(＝NR ^e31 )NR ^c31 C(O)OR ^a31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

wherein each R is ^Cy3C Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy3C The ring atoms of the 5-10 membered heteroaryl or 4-10 membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; wherein R is formed ^Cy3C Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O) ^Rb31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo; and wherein R is formed ^Cy3C Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a31 、SR ^a31 、C(O)R ^b31 、C(O)NR ^c31 R ^d31 、C(O)OR ^a31 、OC(O)R ^b31 、OC(O)NR ^c31 R ^d31 、NR ^c31 R ^d31 、NR ^c31 C(O) ^Rb31 、NR ^c31 C(O)NR ^c31 R ^d31 、NR ^c31 C(O)OR ^a31 、C(＝NR ^e31 )NR ^c31 R ^d31 、NR ^c31 C(＝NR ^e31 )NR ^c31 R ^d31 、S(O)R ^b31 、S(O)NR ^c31 R ^d31 、S(O) ₂ R ^b31 、NR ^c31 S(O) ₂ R ^b31 、S(O) ₂ NR ^c31 R ^d31 And oxo;

R ³⁶ selected from H and C _1-6 An alkyl group;

R ^a31 、R ^b31 、R ^c31 and R is ^d31 Each independently selected from H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a31 、R ^b31 、R ^c31 And R is ^d31 Is not less than C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, halogen, CN, OR ^a32 、SR ^a32 、C(O)R ^b32 、C(O)NR ^c32 R ^d32 、C(O)OR ^a32 、OC(O)R ^b32 、OC(O)NR ^c32 R ^d32 、NR ^c32 R ^d32 、NR ^c32 C(O)R ^b32 、NR ^c32 C(O)NR ^c32 R ^d32 、NR ^c32 C(O)OR ^a32 、C(＝NR ^e32 )NR ^c32 R ^d32 、NR ^c32 C(＝NR ^e32 )NR ^c32 R ^d32 、S(O)R ^b32 、S(O)NR ^c32 R ^d32 、S(O) ₂ R ^b32 、NR ^c32 S(O) ₂ R ^b32 、S(O) ₂ NR ^c32 R ^d32 And oxo;

or R attached to the same N atom ^c31 And R is ^d31 Together with the N atom to which they are both attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: c (C) _1-6 Alkyl, halogen, CN, OR ^a32 、SR ^a32 、C(O)R ^b32 、C(O)NR ^c32 R ^d32 、C(O)OR ^a32 、OC(O)R ^b32 、OC(O)NR ^c32 R ^d32 、NR ^c32 R ^d32 、NR ^c32 C(O)R ^b32 、NR ^c32 C(O)NR ^c32 R ^d32 、NR ^c32 C(O)OR ^a32 、C(＝NR ^e32 )NR ^c32 R ^d32 、NR ^c32 C(＝NR ^e32 )NR ^c32 R ^d32 、S(O)R ^b32 、S(O)NR ^c32 R ^d32 、S(O) ₂ R ^b32 、NR ^c32 S(O) ₂ R ^b32 、S(O) ₂ NR ^c32 R ^d32 And oxo;

R ^a32 、R ^b32 、R ^c32 and R is ^d32 Each independently selected from H, C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a32 、R ^b32 、R ^c32 And R is ^d32 Is not less than C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

or R attached to the same N atom ^c32 And R is ^d32 Together with the N atom to which they are each attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each of which is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo; and

R ^e31 and Re (Re) ³² Each independently is H, CN or NO ₂ ；

Cy ^4A Is unsubstituted or substituted C _6-10 Aryl, or unsubstituted or substituted 5-10 membered heteroaryl; in which Cy is formed ^4A The ring atoms of the 5-10 membered heteroaryl group consist of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S; in which Cy is formed ^4A Substituted C of (2) _6-10 Aryl or substituted 5-10 membered heteroaryl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy4A Halogen, C _1-6 Haloalkyl, CN, OR ^a41 、S ^Ra41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、C(＝NOR ^a41 )NR ^c41 R ^d41 、C(＝NOC(O)R ^b41 )NR ^c41 R ^d41 、C(＝NR ^e41 )NR ^c41 C(O)OR ^a41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

each R ^Cy4A Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy4A The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, N and S, wherein R is formed ^Cy4A Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo, and wherein R is formed ^Cy4A Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

R ⁴¹ is H or C _1-6 Alkyl, C _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl, wherein R is formed ⁴¹ C of (2) _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo, and wherein R is formed ⁴¹ C of (2) _6-10 aryl-C _1-6 Alkyl or 5-to 10-membered heteroaryl-C _1-6 Alkyl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

R ⁴² h, C of a shape of H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl or Cy ^4B The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is formed ⁴² Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: cy (Cy) ^4B Halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo; provided that no more than one substituent is Cy ^4B ；

Cy ^4B Is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^4B The ring atoms of the 5-10 membered heteroaryl, or unsubstituted or substituted 4-10 membered heterocycloalkyl, consisting of 1, 2 or 3 heteroatoms selected from O, N and S; and wherein Cy is formed ^4B Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy4B Halogen, C _1-6 Haloalkyl, CN, OR ^a41 、S ^Ra41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、C(＝NOR ^a41 )NR ^c41 R ^d41 、C(＝NOC(O)R ^b41 )NR ^c41 R ^d41 、C(＝NR ^e41 )NR ^c41 C(O)OR ^a41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

wherein each R is ^Cy4B Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy4B The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, and wherein R is formed ^Cy4B Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo; and form R ^Cy4B Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

or R is ⁴¹ And R is ⁴² Together with the atoms to which they are attached, and the linkage R ⁴¹ And R is ⁴² Together with the nitrogen atom of the atom to which it is attached, form a 4-7 membered heterocycloalkyl ring; which is optionally further substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy4B Halogen, C _1-6 Haloalkyl, CN, OR ^a41 、S ^Ra41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、C(＝NOR ^a41 )NR ^c41 R ^d41 、C(＝NOC(O)R ^b41 )NR ^c41 R ^d41 、C(＝NR ^e41 )NR ^c41 C(O)OR ^a41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

R ⁴³ h, C of a shape of H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl or Cy ^4C The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is formed ⁴³ Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents each independently selected from: 0, 1, 2, 3, 4 or 5 substituents selected from: cy (Cy) ^4C Halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo, provided that R is formed ⁴³ C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 No more than one substituent of alkynyl is Cy ^4C ；

Cy ^4C Is unsubstituted or substituted C _6-10 Aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C _3-10 Cycloalkyl, or unsubstituted or substituted 4-10 membered heterocycloalkyl; in which Cy is formed ^4B The ring atoms of the 5-10 membered heteroaryl, or unsubstituted or substituted 4-10 membered heterocycloalkyl, consisting of 1, 2 or 3 heteroatoms selected from O, N and S; and wherein Cy is formed ^4C Substituted C of (2) _6-10 Aryl, substituted 5-10 membered heteroaryl, substituted C _3-10 Cycloalkyl or 4-10 membered heterocycloalkyl is substituted with 1, 2, 3, 4 or 5 substituents each independently selected from R ^Cy4C Halogen, C _1-6 Haloalkyl, CN, OR ^a41 、S ^Ra41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、C(＝NOR ^a41 )NR ^c41 R ^d41 、C(＝NOC(O)R ^b41 )NR ^c41 R ^d41 、C(＝NR ^e41 )NR ^c41 C(O)OR ^a41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

each R ^Cy4C Independently selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl, wherein R is formed ^Cy4C The ring atoms of the 5-to 10-membered heteroaryl or 4-to 10-membered heterocycloalkyl group consisting of carbon atoms and 1, 2 or 3 heteroatoms selected from O, N and S, wherein R is formed ^Cy4C Each C of (2) _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo; and wherein each R is formed ^Cy4A Each C of (2) _6-10 Aryl, 5-10 membered heteroaryl, C _3-10 Cycloalkyl and 4-10 membered heterocycloalkyl are independently unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: halogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, CN, OR ^a41 、SR ^a41 、C(O)R ^b41 、C(O)NR ^c41 R ^d41 、C(O)OR ^a41 、OC(O)R ^b41 、OC(O)NR ^c41 R ^d41 、NR ^c41 R ^d41 、NR ^c41 C(O)R ^b41 、NR ^c41 C(O)NR ^c41 R ^d41 、NR ^c41 C(O)OR ^a41 、C(＝NR ^e41 )NR ^c41 R ^d41 、NR ^c41 C(＝NR ^e41 )NR ^c41 R ^d41 、S(O)R ^b41 、S(O)NR ^c41 R ^d41 、S(O) ₂ R ^b41 、NR ^c41 S(O) ₂ R ^b41 、S(O) ₂ NR ^c41 R ^d41 And oxo;

R ^a41 、R ^b41 、R ^c41 and R is ^d41 Each independently selected from H, C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a41 、R ^b41 、R ^c41 And R is ^d41 Is not less than C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _6-10 Aryl, C _3-7 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C _6-10 aryl-C _1-3 Alkyl, 5-10 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-10 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: c (C) _1-6 Alkyl, halogen, CN, OR ^a42 、SR ^a42 、C(O)R ^b42 、C(O)NR ^c42 R ^d42 、C(O)OR ^a42 、OC(O)R ^b42 、OC(O)NR ^c42 R ^d42 、NR ^c42 R ^d42 、NR ^c42 C(O)R ^b42 、NR ^c42 C(O)NR ^c42 R ^d42 、NR ^c42 C(O)OR ^a42 、C(＝NR ^e42 )NR ^c42 R ^d42 、NR ^c42 C(＝NR ^e42 )NR ^c42 R ^d42 、S(O)R ^b42 、S(O)NR ^c42 R ^d42 、S(O) ₂ R ^b42 、NR ^c42 S(O) ₂ R ^b42 、S(O) ₂ NR ^c42 R ^d42 And oxo;

or R attached to the same N atom ^c41 And R is ^d41 Together with the N atom to which they are both attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each optionally substituted with 1, 2 or 3 substituents independently selected from: c (C) _1-6 Alkyl, halogen, CN, OR ^a42 、SR ^a42 、C(O)R ^b42 、C(O)NR ^c42 R ^d42 、C(O)OR ^a42 、OC(O)R ^b42 、OC(O)NR ^c42 R ^d42 、NR ^c42 R ^d42 、NR ^c42 C(O)R ^b42 、NR ^c42 C(O)NR ^c42 R ^d42 、NR ^c42 C(O)OR ^a42 、C(＝NR ^e42 )NR ^c42 R ^d42 、NR ^c42 C(＝NR ^e42 )NR ^c42 R ^d42 、S(O)R ^b42 、S(O)NR ^c42 R ^d42 、S(O) ₂ R ^b42 、NR ^c42 S(O) ₂ R ^b42 、S(O) ₂ NR ^c42 R ^d42 And oxo;

R ^a42 、R ^b42 、R ^c42 and R is ^d42 Each independently selected from H, C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, wherein R is formed ^a42 、R ^b42 、R ^c42 And R is ^d42 Is not less than C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, phenyl, C _3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C _1-3 Alkyl, 5-6 membered heteroaryl-C _1-3 Alkyl, C _3-7 cycloalkyl-C _1-3 Alkyl and 4-7 membered heterocycloalkyl-C _1-3 Alkyl, each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo;

or R attached to the same N atom ^c42 And R is ^d42 Together with the N atom to which they are each attached, form a 4, 5, 6 or 7 membered heterocycloalkyl or 5 membered heteroaryl, each of which is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from: OH, CN, amino, NH (C) _1-6 Alkyl), N (C) _1-6 Alkyl group ₂ Halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, C _1-6 Haloalkoxy and oxo; and

R ^e41 and Re (Re) ⁴² Each independently is H, CN or NO ₂ 。

In some embodiments, the small molecule is of compound formula (VA) or (VB):

or a salt thereof;

wherein:

A ¹ is selected from- (c=nh) -, - (c=nor) ^a )–、–[C＝NO(C＝O)R ^a ]–、–[C＝N[O(C＝O)ZR ^b ]A member of the group consisting of a } -, a fused 5-or 6-membered heterocyclyl and a fused 5-or 6-membered heteroaryl;

when A is ¹ In the case of- (c=nh) -Y ¹ Selected from the group consisting of-NH ₂ 、–NH(C＝O)R ^a and-NH (c=o) ZR ^b ；

When A is ¹ Is- (c=nor) ^a )–、–[C＝NO(C＝O)R ^a ]-or- { c=n [ O (c=o) ZR ^b ]When } is } -Y ¹ is-NH ₂ ；

When A is ¹ Is a condensed heterocyclic group orIn the case of heteroaryl groups, Y ¹ is-NH ₂ Or halogen, and A ¹ From m further R ¹ Group substitution;

each R ^a And R is ^b Independently selected from C ₁ -C ₆ Alkyl, C ₃ -C ₁₀ Cycloalkyl, C ₆ -C ₁₀ Aryl and C ₇ -C ₁₂ An arylalkyl group; wherein R is ^a Having m substituents selected from the group consisting of: c (C) ₁ -C ₆ Alkyl, hydroxy (C) ₁ -C ₆ Alkyl group, C ₁ -C ₆ Alkoxy, C ₂ -C ₉ Alkoxyalkyl, amino, C ₁ -C ₆ Alkylamino and halogen; or, alternatively, R ^a And R is ^b Ligating to form a mixture having a structure selected from C ₁ -C ₆ Alkyl, hydroxy, C ₁ -C ₆ Heterocyclyl rings of m substituents for alkoxy and halogen;

each Z is independently selected from O and S;

A ² is selected from C ₃ -C ₆ Heteroaryl, C ₆ Aryl and C ₂ -C ₆ A member of the alkyl group;

when A is ² Is C ₃ -C ₆ In the case of heteroaryl groups, Y ² Selected from the group consisting of-NH ₂ 、–CH ₂ NH ₂ Chlorine, - (c=nh) NH ₂ 、–(C＝NH)NH(C＝O)R ^a 、–(C＝NH)NH(C＝O)ZR ^b 、–(C＝NOR ^a )NH ₂ 、–[C＝NO(C＝O)R ^a ]NH ₂ And- { c=n [ O (c=o) ZR ^b ]}NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And A is ² From m further R ¹ Group substitution;

when A is ² Is C ₆ In the case of aryl radicals, Y ² Selected from aminomethyl, hydroxy and halogen, and A ² From m further R ¹ Group substitution;

when A is ² Is C ₂ -C ₆ In the case of alkyl radicals, Y ² Selected from-NH (c=nh) NH ₂ 、–NH(C＝NH)NH(C＝O)R ^a and-NH (c=nh) NH (c=o) ZR ^b ；

Each R ¹ Is independent ofIs selected from C ₁ -C ₆ Alkyl, hydroxy, C ₁ -C ₆ Alkoxy, amino, C ₁ -C ₆ Alkylamino and halogen members;

each m and n is an integer independently selected from 0 to 3;

l is- (O) _p –(C(R ^2a )(R ^2b )) _q –，

Each R ^2a Or R is ^2b Is a member independently selected from hydrogen and fluorine;

p is an integer from 0 to 1;

q is an integer from 1 to 2;

R ³ is selected from hydrogen, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Fluoroalkyl and carboxyl (C) ₁ -C ₆ Alkyl); or, alternatively, R ³ And R is ⁴ To form an azetidine, pyrrolidine or piperidine ring; or (b)

R ⁴ Is selected from hydrogen and C ₁ -C ₆ A member of the alkyl group; or, alternatively, R ³ And R is ⁴ To form an azetidine, pyrrolidine or piperidine ring;

R ⁵ is selected from C ₃ -C ₇ Cycloalkyl, C ₄ -C ₈ Cycloalkylalkyl, heteroaryl and having 0 to 3R ¹³ C of substituents ₇ -C ₁₂ A member of the arylalkyl or heteroarylalkyl group; or, alternatively, R ⁵ And R is ⁶ Ligating to form a compound having 0 to 3R ¹³ Heterocyclic ring of substituent;

R ⁶ is selected from hydrogen, C ₁ -C ₆ Alkyl, C ₃ -C ₇ Cycloalkyl, carboxyl (C) ₁ -C ₆ Alkyl), having 0 to 3R ¹³ C of substituents ₇ -C ₁₂ Arylalkyl or heteroarylalkyl, amino (C) ₁ -C ₈ An alkyl group); and an amido (C) ₁ -C ₈ Alkyl); or, alternatively, R ⁵ And R is ⁶ Ligating to form a compound having 0 to 3R ¹³ Heterocyclic ring of substituent; and

each of which isR ¹³ Is independently selected from C ₁ -C ₆ Alkyl, C ₆ -C ₁₀ Aryl, (C) ₆ -C ₁₀ Aryl) C ₁ -C ₆ Alkyl, carboxyl (C) ₁ -C ₆ Alkoxy), heteroaryl, (C ₆ -C ₁₀ Heteroaryl) C ₁ -C ₆ Alkyl, heterocyclyl, hydroxy (C) ₁ -C ₆ Alkyl group, C ₁ -C ₆ Alkoxy, C ₂ -C ₉ Alkoxyalkyl, amino, C ₁ -C ₆ Amido, C ₁ -C ₆ Alkylamino and halogen members; or, alternatively, two R ¹³ The groups being linked to form a condensed C ₆ -C ₁₀ Aryl, C ₆ -C ₁₀ Heteroaryl or C ₅ -C ₇ Cycloalkyl rings.

In some embodiments, the small molecule is a compound of formula (VIA) or (VIB):

Or a salt thereof; wherein:

A ¹ is selected from- (c=nh) -, - (c=nor) ^a )–、–[C＝NO(C＝O)R ^a ]–、–[C＝N[O(C＝O)ZR ^b ]]-a member of a fused 5 or 6 membered heterocyclyl and fused 5 or 6 membered heteroaryl;

When A is ¹ In the case of fused heterocyclyl or heteroaryl groups, Y ¹ is-NH ₂ Or halogen, and A ¹ From m further R ¹ Group substitution;

each Z is independently selected from O and S;

A ² is selected from C ₃ -C ₆ Heteroaryl and C ₂ -C ₆ A member of the alkyl group;

Each R ¹ Is independently selected from C ₁ -C ₆ Alkyl, hydroxy, C ₁ -C ₆ Alkoxy, amino, C ₁ -C ₆ Alkylamino and halogen members;

each m and n is an integer independently selected from 0 to 3;

x and X ² Each is selected from NR ⁸ CH and CR ¹⁰ Is a member of (2);

each R ⁸ Is independently selected from hydrogen and C ₁ -C ₆ A member of the alkyl group;

each R ¹⁰ Is independently selected from C ₁ -C ₆ Alkyl, having 0 to 3R ¹³ Heteroaryl or C of substituents ₆ -C ₁₀ Aryl, hydroxy (C) ₁ -C ₆ Alkyl group, C ₁ -C ₆ Alkoxy, C ₂ -C ₉ Alkoxyalkyl, amino, C ₁ -C ₆ Alkylamino and halogen members; or, alternatively, two R ¹⁰ The radicals being linked to form a chain having 0 to 3R ¹³ Condensed C of substituents ₆ Aryl, heteroaryl or C ₅ -C ₇ A cycloalkyl ring; or (b)

r is an integer from 0 to 4; and

each R ¹³ Is independently selected from C ₁ -C ₆ Alkyl, C ₆ -C ₁₀ Aryl, carboxyl (C) ₁ -C ₆ Alkoxy), heteroaryl, heterocyclyl, hydroxy (C ₁ -C ₆ Alkyl group, C ₁ -C ₆ Alkoxy, C ₂ -C ₉ Alkoxyalkyl, amino, C ₁ -C ₆ Amido, C ₁ -C ₆ Alkylamino and halogen members; or, alternatively, two R ¹³ The groups being linked to form a condensed C ₆ -C ₁₀ Aryl, C ₆ -C ₁₀ Heteroaryl or C ₅ -C ₇ Cycloalkyl rings.

In certain embodiments, the small molecule is a compound of formula (VIIA) or (VIIB):

or a salt thereof;

wherein:

each Z is independently selected from O and S;

when A is ² Is C ₂ -C ₆ In the case of an alkyl group, the alkyl group,Y ² selected from-NH (c=nh) NH ₂ 、–NH(C＝NH)NH(C＝O)R ^a and-NH (c=nh) NH (c=o) ZR ^b ；

each m and n is an integer independently selected from 0 to 3;

l is- (O) _p –(C(R ^2a )(R ^2b )) _q –，

p is an integer from 0 to 1;

q is an integer from 1 to 2;

R ³ is selected from hydrogen, C ₁ -C ₆ Alkyl and carboxyl (C) ₁ -C ₆ Alkyl);

each R ¹¹ Is independently selected from C ₁ -C ₆ Alkyl, hydroxy, C ₁ -C ₆ Alkoxy, amino, C ₁ -C ₆ Alkylamino, halo and (R) ¹⁴ )(R ¹⁴ ) A member of N (CO) -; or, alternatively, two R ¹¹ The radicals being linked to form a chain having 0 to 3R ¹³ Condensed C of substituents ₆ Aryl, heteroaryl or C ₅ -C ₇ A cycloalkyl ring; or (b)

r is an integer from 0 to 4; and

each Z is independently selected from O and NR ⁸ Is a member of (2);

each R ¹² Is independently selected from hydrogen, having 0 to 3R ¹³ C of substituents ₁ -C ₆ Alkyl and C ₇ -C ₁₄ A member of the arylalkyl group;

each R ¹³ Is independently selected from C ₁ -C ₆ Alkyl, hydroxy (C) ₁ -C ₆ Alkyl group, C ₁ -C ₆ Alkoxy, C ₂ -C ₉ Alkoxyalkyl, amino, C ₁ -C ₆ Alkylamino and halogen members; or, alternatively, two R ¹³ The groups being linked to form a condensed C ₆ Aryl, heteroaryl or C ₅ -C ₇ A cycloalkyl ring; and

each R ¹⁴ Is independently selected from hydrogen, C ₁ -C ₆ Alkyl, C ₃ -C ₇ Cycloalkyl, C ₄ -C ₈ Cycloalkylalkyl, C ₇ -C ₁₄ Arylalkyl and heteroaryl (C) ₁ -C ₆ Alkyl); or, alternatively, two R ¹³ The groups are linked to form a fused heterocyclyl ring.

In some embodiments, the small molecule is a compound having the structure:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

R ¹ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl;

R ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ or R is ^2j Independently selected from hydrogen, halogen, C (=o) OR ⁵ 、OC(＝O)R ⁵ Hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR ⁵ R ⁶ 、C(＝O)NR ⁵ R ⁶ 、N(R ⁵ )C(＝O)R ⁶ 、NR ⁵ C(＝O)NR ⁶ 、S(O) _t 、SR ⁵ Nitro, N (R) ⁵ )C(O)OR ⁶ 、C(＝NR ⁵ )NR ⁶ R ⁷ 、N(R ⁵ )C(＝NR ⁶ )NR ⁷ R ⁸ 、S(O)R ⁵ 、S(O)NR ⁵ R ⁶ 、S(O) ₂ R ⁵ 、N(R ⁵ )S(O) ₂ R ⁶ 、S(O) ₂ NR ⁵ R ⁶ Aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ Or R is ^2j Is not hydrogen;

R ³ is NR (NR) ^3a R ^3b ；

R ^3a And R is ^3b Each independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH) ₂ ) _n C(＝O)OR ⁶ Or (CH) ₂ ) _n P(＝O)(OR ⁶ ) ₂ ；

Or R is ^3a And R is ^3b Together with the nitrogen to which they are attached, form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

Or R is ^3a And R is ⁴ Together with the nitrogen and carbon to which they are attached, form an optionally substituted 4-7 membered heterocyclyl;

when n is 2, 3, 4, 5 or 6, R ⁴ Is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl; or (b)

When n is 0 or 1, R ⁴ Is a substituted or unsubstituted monocyclic heteroaryl, or a substituted or unsubstituted heterocyclyl;

at each occurrence, R ⁵ 、R ⁶ 、R ⁷ And R is ⁸ Independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

x is a direct bond, -CR ^2e R ^2f -or-CR ^2e R ^2f -CR ^2g R ^2h -；

Y is a direct bond or-CR ²ⁱ R ^2j -；

n is an integer of 0 to 6; and

t is 1-3.

In some embodiments, the small molecule is a compound having the structure:

R ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ or R is ^2j Independently selected from hydrogen, halogen, OR ⁵ 、C(＝O)OR ⁵ 、OC(＝O)R ⁵ Hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR ⁵ R ⁶ 、C(＝O)NR ⁵ R ⁶ 、N(R ⁵ )C(＝O)R ⁶ 、NR ⁵ C(＝O)NR ⁶ 、S(O) _t 、SR ⁵ Nitro, N (R) ⁵ )C(O)OR ⁶ 、C(＝NR ⁵ )NR ⁶ R ⁷ 、N(R ⁵ )C(＝NR ⁶ )NR ⁷ R ⁸ 、S(O)R ⁵ 、S(O)NR ⁵ R ⁶ 、S(O) ₂ R ⁵ 、N(R ⁵ )S(O) ₂ R ⁶ 、S(O) ₂ NR ⁵ R ⁶ Aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ Or R is ^2j Is not hydrogen;

R ³ is NR (NR) ^3a R ^3b ；

R ^3a And R is ^3b Each independently is hydrogen, alkyl,Hydroxyalkyl, haloalkyl, alkoxyalkyl, -CH ₂ C(C＝O)OH、-CH ₂ C (=o) O alkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl or cycloalkyl;

x is a direct bond, - [ C (R) ^2e )R ^2f ]-or- [ C (R) ^2e )R ^2f ]-[C(R ^2g )R ^2h ]-；

Y is a direct bond or- [ C (R) ²ⁱ )R ^2j ]-；

n is an integer of 0 to 6; and

the t is 1 to 3 and the total number of the components is,

the conditions are as follows:

a) When R is ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ Or R is ^2j When one occurrence of (2) is OH, R ¹ The structure is not as follows:

b) When R is ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g Or R is ^2h When one occurrence of (C) is-OH, n is an integer of 2 to 6A number; and

c) When R is ^2a 、R ^2b 、R ^2c 、R ^2d 、R ^2e 、R ^2f 、R ^2g 、R ^2h 、R ²ⁱ Or R is ^2j R when one occurrence is unsubstituted phenyl ^3a And R is ^3b None of the following structures:

in some embodiments, the small molecule is a compound having the structure:

R ¹⁷ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl;

R ^18a 、R ^18b 、R ^18c 、R ^18d 、R ^18e 、R ^18f 、R ^18g 、R ^18h 、R ¹⁸ⁱ or R is ^18j Independently selected from hydrogen, halogen, -OR ²¹ 、C(＝O)OR ²¹ 、OC(＝O)R ²¹ Hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR ²¹ R ²² 、C(＝O)NR ²¹ R ²² 、N(R ²¹ )C(＝O)R ²² 、NR ²¹ C(＝O)NR ²² 、S(O) _t 、SR ²¹ Nitro, N (R) ²¹ )C(O)OR ²² 、C(＝NR ²¹ )NR ²² R ²³ 、N(R ²¹ )C(＝NR ²² )NR ²³ R ²⁴ 、S(O)R ²¹ 、S(O)NR ²¹ R ²² 、S(O) ₂ R ²¹ 、N(R ²¹ )S(O) ₂ R ²² 、S(O) ₂ NR ²¹ R ²² Aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R ^18a 、R ^18b 、R ^18c 、R ^18d 、R ^18e 、R ^18f 、R ^18g 、R ^18h 、R ¹⁸ⁱ Or R is ^18j Is not hydrogen;

R ¹⁹ is NR (NR) ^19a R ^19b ；

R ^19a And R is ^19b Each independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH) ₂ ) _n C(＝O)OR ⁵ Or (CH) ₂ ) _n P(＝O)(OR ⁵ ) ₂ ；

Or R is ^19a And R is ^19b Together with the nitrogen to which they are attached, form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

R ²⁰ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl;

At each occurrence, R ²¹ 、R ²² 、R ²³ And R is ²⁴ Independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

x is a direct bond, -CR ^2e R ^2f -or-CR ^2e R ^2f -CR ^2g R ^2h -；

Y is a direct bond or-CR ²ⁱ R ^2j -；

Z is O or S;

m is an integer of 0 to 6; and

t is 1-3.

In certain embodiments, the small molecule is a compound having the structure:

R ²⁵ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl;

R ^26a 、R ^26b 、R ^26c or R is ^26d Independently selected from hydrogen, halogen, -OR ²⁹ 、C(＝O)OR ²⁹ 、OC(＝O)R ²⁹ Hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkoxy, cyano, aminoalkyl, carboxyalkyl, NR ²⁹ R ³⁰ 、C(＝O)NR ²⁹ R ³⁰ 、N(R ²⁹ )C(＝O)R ³⁰ 、NR ²⁹ C(＝O)NR ³⁰ 、S(O) _t 、SR ²⁹ Nitro, N (R) ²⁹ )C(O)OR ³⁰ 、C(＝NR ²⁹ )NR ³⁰ R ³¹ 、N(R ²⁹ )C(＝NR ³⁰ )NR ³¹ R ³² 、S(O)R ²⁹ 、S(O)NR ²⁹ R ³⁰ 、S(O) ₂ R ³⁰ 、N(R ²⁹ )S(O) ₂ R ³⁰ 、S(O) ₂ NR ²⁹ R ³⁰ Aryl, heteroaryl, heterocyclyl, cycloalkyl and oxo, provided that R ^26a 、R ^26b 、R ^26c Or R is ^26d Is not hydrogen;

R ²⁷ is NR (NR) ^27a R ^27b ；

R ^27a And R is ^27b Each independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, (CH) ₂ ) _n C(＝O)OR ²⁹ Or (CH) ₂ ) _n P(＝O)(OR ²⁹ ) ₂ ；

Or R is ^27a And R is ^27b Together with the nitrogen to which they are attached, form an optionally substituted 4-7 membered heteroaryl or an optionally substituted 4-7 membered heterocyclyl;

R ²⁸ Is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstitutedOr a substituted or unsubstituted heterocyclic group;

at each occurrence, R ²⁹ 、R ³⁰ 、R ³¹ And R is ³² Independently is hydrogen, alkyl, hydroxyalkyl, haloalkyl, alkoxyalkyl, carboxyalkyl, heterocyclyl, heteroaryl, or cycloalkyl;

x is a direct bond or-CR ^26c R ^26d -；

p is an integer of 0 to 6; and

t is 1-3.

In some embodiments, the small molecule is a compound having the structure:

R ² is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

R ³ hydrogen, alkyl, haloalkyl or cycloalkyl;

or R is ² And R is ³ Together with the carbon and nitrogen to which they are attached, form an optionally substituted 4-7 membered heterocyclyl;

R ⁴ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl;

R ^5a is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphanyl, (CH) ₂ ) _n C(＝O)OR ⁶ 、C(＝O)R ⁶ 、C(＝O)OR ⁶ Or C (=O) NR ⁶ R ⁷ ；

R ^5b Is electron pair or alkyl;

at each occurrence, R ⁶ And R is ⁷ Independently hydrogen, alkyl, haloalkyl, cycloalkyl or arylalkyl;

R ⁸ is alkyl, haloalkyl, aminoalkyl, substituted or unsubstituted arylalkyl; and

n is 1, 2, 3, 4, 5, 6, 7 or 8,

provided that it is

A)R ^5a Is alkyl, haloalkyl, cycloalkyl, phosphinoalkyl, (CH) ₂ ) _n C(＝O)OR ⁶ 、C(＝O)R ⁶ 、C(＝O)OR ⁶ Or C (=O) NR ⁶ R ⁷ Or R is ¹ From substituted heteroaryl groups, C (=nh) NHC (=o) OR ⁸ 、C(＝NOC(＝O)R ⁸ )NH ₂ 、C(＝NOC(＝O)OR ⁸ )NH ₂ And C (=NOH) NH ₂ Is substituted with one or more substituents; and

b) When R is ^5a Is alkyl or (CH) ₂ ) _n C(＝O)OR ⁶ When R is ¹ The structure is not as follows:

unless R is ² And R is ³ Together with the carbon and nitrogen to which they are attached, form an optionally substituted 4-7 membered heterocyclyl, respectively.

In some embodiments, the small molecule is a compound having the structure:

R ² is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

R ³ hydrogen, alkyl, haloalkyl or cycloalkyl;

R ^5b Is electron pair or alkyl;

n is 1, 2, 3, 4, 5, 6, 7 or 8,

provided that it is

b) The compound of structure (I) does not have one of the following structures:

in some embodiments, the small molecule is a compound having the structure:

R ² Is hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy or cycloalkyl;

R ³ hydrogen, alkyl, haloalkyl or cycloalkyl;

at each occurrence, R ^5a And R is ^5b Independently having one of the following structures:

or R is ^5a And R is ^5b Together with the phosphorus atom to which they are attached, form an optionally substituted 4-7 membered heterocyclyl;

R ^6a is alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;

at each occurrence, R ^6b Independently hydrogen or alkyl;

at each occurrence, R ⁷ Independently alkyl, haloalkyl, heteroaryl, cycloalkyl, and heterogenousA cyclic, arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocyclylalkyl group;

R ⁸ is an amino acid side chain; and

n is 1, 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the small molecule is a compound having the structure:

Represents a double bond or a single bond;

R ¹ is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

R ² is hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, haloalkoxy or cycloalkyl;

R ³ is hydrogen, alkyl, haloalkyl or cycloalkyl, or R ² And R is ³ Together with the carbon and nitrogen to which they are attached, form an optionally substituted 4-7 membered heterocyclyl;

R ⁵ is hydrogen, alkyl, haloalkyl, cycloalkyl, phosphanyl, (CH) ₂ ) _m C(＝O)OR ⁶ 、C(＝O)R ⁶ 、C(＝O)OR ⁶ 、(CH ₂ ) _m NR ⁶ S(O) ₂ R ⁷ Or C (=O) NR ⁶ R ⁷ ；

L ¹ is a direct bond, -CR ^8a R ^8b -、-S(O) _t -、NR ^8c or-O-;

R ^8a and R is ^8b Each independently is hydrogen, alkyl, or R ^8a And R is ^8b Together with the carbon to which they are attached, form an optionally substituted 3-6 membered cycloalkyl;

R ^8c is hydrogen, alkyl, haloalkyl, (c=o) alkyl, (c=o) oalkyl, (c=o) cycloalkyl, (c=o) oaycloalkyl, (c=o) aryl, (c=o) oaryl, (c=o) heteroaryl, (c=o) oaheteroaryl, (c=o) heterocyclyl, (c=o) O heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl;

n is 1 or 2;

m is 1, 2, 3, 4, 5 or 6; and

t is 0, 1 or 2.

In some embodiments, the small molecule is a compound having the structure:

R ¹ is a substituted or unsubstituted heteroaryl;

R ² is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

R ³ is hydrogen or alkyl;

R ⁴ is alkyl, substituted or unsubstituted arylalkyl, heterocyclyl substituted with a substituent selected from substituted or unsubstituted phenyl or substituted or unsubstituted pyridinyl, or R ³ And R is ⁴ Together with the nitrogen and carbon to which they are attached, form an optionally substituted 4-10 membered heterocyclyl;

R ^5a hydrogen or halogen;

R ^5b is hydrogen, alkyl, haloalkyl, (c=o) alkyl, (c=o) oalkyl, (c=o) cycloalkyl, (c=o) oaycloalkyl, (c=o) aryl, (c=o) oaryl, (c=o) heteroaryl, (c=o) oaheteroaryl, (c=o) heterocyclyl, (c=o) O heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl;

L ¹ Is a direct bond, -CH ₂ -、-S(O) _t -、NR ^5b -O-, -c=c-, or-c≡c-; and

t is 0, 1 or 2,

the conditions are as follows:

A)R ² does not have one of the following structures:

B)R ¹ does not have one of the following structures:

and

c) When R is ² In the case of unsubstituted phenyl, R ¹ Does not have one of the following structures:

in certain more specific embodiments, the small molecule is a compound having the structure:

R ⁶ is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

R ⁷ is alkyl, -SR ¹⁰ Substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R ⁸ hydrogen, alkyl, haloalkyl or cycloalkyl;

R ⁹ is a substituted or unsubstituted arylalkyl, a substituted or unsubstituted heteroarylalkyl, or R ⁸ And R is ⁹ Together with the nitrogen to which they are attached, form an optionally substituted 4-10 membered heterocyclyl;

R ¹⁰ hydrogen, alkyl, haloalkyl or cycloalkyl;

the conditions are as follows:

a) When R is ⁷ Is unsubstituted phenyl, 3- ((methylsulfonyl) amino) phenyl, 2-methylphenyl, 3- (dimethylamino) phenyl, 3- (methylamino) phenyl, 3-methylphenyl, 3-aminomethylphenyl, 3-aminophenyl, unsubstituted pyridyl, 3- (methylamino) -2-thienyl, 3, 4-diamino-2-thienyl, 3- ((methylsulfonyl) amino) -2-thienyl, 3-amino-5-5 (aminocarbonyl) phenyl, or one of the following structures:

R ⁶ The structure is not as follows:

b) When R is ⁷ In the case of unsubstituted phenyl, R ⁶ The structure is not as follows:

in some embodiments, the small molecule is a compound having the structure:

R ¹¹ has one of the following structures:

R ¹² methyl or halogen;

R ¹³ is a substituted or unsubstituted aryl group; and

n is 1 or 2

The conditions are as follows:

the compound of structure (III) does not have the following structure:

in some more specific embodiments, the small molecule is a compound having the structure:

R ¹⁴ is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

R ¹⁵ is a substituted or unsubstituted arylalkyl, or a substituted or unsubstituted heteroarylalkyl;

L ² is a direct bond, -C (=O) or-S (=O) _t -; and

t is 0, 1 or 2.

Inhibitors of MASP-2 expression

In another embodiment of this aspect of the invention, the MASP-2 inhibitor is an inhibitor of MASP-2 expression that is capable of inhibiting MASP-2 dependent complement activation. In the practice of this aspect of the invention, representative inhibitors of MASP-2 expression include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2RNAi molecules.

Antisense RNA and DNA molecules act to directly block translation of MASP-2mRNA by hybridizing to MASP-2mRNA and preventing translation of MASP-2 protein. The antisense nucleic acid molecule can be constructed in a number of different ways, provided that it is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by reversing the coding region (or a portion thereof) of MASP-2cDNA (SEQ ID NO: 4) relative to its normal direction of transcription to allow transcription of its complement.

The antisense nucleic acid molecules are generally substantially identical to at least a portion of one or more target genes. However, the nucleic acids need not be identical to inhibit expression. Generally, higher homology can be used to compensate for the use of shorter antisense nucleic acid molecules. The minimal percent identity is typically greater than about 65%, but higher percent identity may exert more effective repression of expression of the endogenous sequence. A substantially higher percentage of identity of greater than about 80% is generally preferred, although about 95% to absolute identity is generally most preferred.

The antisense nucleic acid molecule need not have the same intron or exon pattern as the target gene, and the non-coding segment of the target gene can be as effective as the coding segment in achieving antisense suppression of target gene expression. DNA sequences of at least about 8 nucleotides or so may be used as antisense nucleic acid molecules, although longer sequences are preferred. In the present invention, a representative example of a useful MASP-2 inhibitor is an antisense MASP-2 nucleic acid molecule that is at least ninety percent identical to the complement of MASP-2cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO. 4. The nucleic acid sequence shown in SEQ ID NO. 4 encodes a MASP-2 protein consisting of the amino acid sequence shown in SEQ ID NO. 5.

Targeting of antisense oligonucleotides to MASP-2mRNA is another mechanism that may be used to reduce the level of MASP-2 protein synthesis. For example, synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. patent No. 5,739,119 to Cheng and U.S. patent No. 5,759,829 to Shewmaker). Furthermore, examples of antisense inhibition have been demonstrated with the following: nucleoprotein cyclin, multidrug resistance gene (MDG 1), ICAM-1, E-selectin, STK-1, striatal GABA _A Receptor and human EGF (see, e.g., U.S. patent No. 5,801,154 to Baracchini, U.S. patent No. 5,789,573 to Baker, U.S. patent No. 5,718,709 to condidine, and U.S. patent No. 5,610,288 to Reubenstein).

Systems have been described that allow one of ordinary skill to determine which oligonucleotides can be used in the present invention that involve the use of RNase H cleavage as an indicator of sequence accessibility within a transcript to detect a suitable site in a target mRNA. Scherr, M.et al, nucleic Acids Res.26:5079-5085, 1998; lloyd et al, nucleic Acids Res.29:3665-3673, 2001. A mixture of antisense oligonucleotides complementary to certain regions of MASP-2 transcripts is added to a MASP-2 expressing cell extract, such as hepatocytes, and hybridization is performed to create sites susceptible to RNase H. This approach can be combined with computer-assisted sequence selection that can predict optimal sequence selection for antisense compositions based on its relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in a host cell. These secondary structural analysis and target site selection considerations can be performed using OLIGO primer analysis software (Rychlik, i., 1997), and BLASTN 2.0.5 algorithm software (Altschul, s.f., et al, nucleic acids res.25:3389-3402, 1997). The antisense compound directed against the target sequence preferably comprises a length of about 8 to about 50 nucleotides. Antisense oligonucleotides comprising about 9 to about 35 nucleotides are particularly preferred. The inventors contemplate that all oligonucleotide compositions in the range of 9 to 35 nucleotides (i.e., compositions of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases in length) are highly preferred for the practice of the antisense oligonucleotide-based methods of the invention. Highly preferred target regions of MASP-2mRNA are those at or near the AUG translation initiation codon, as well as sequences which are substantially complementary to the 5' region of the mRNA, for example the-10 to +10 region of the nucleotide sequence of the MASP-2 gene (SEQ ID NO: 4). Exemplary inhibitors of MASP-2 expression are provided in Table 4.

Table 4: exemplary inhibitors of MASP-2 expression

As noted above, the term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimics thereof. The term also encompasses oligonucleotide bases consisting of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring modifications. These modifications allow the introduction of certain desirable properties not provided by naturally occurring oligonucleotides, such as reduced toxicity properties, increased stability against nuclease degradation, and enhanced cellular uptake. In an illustrative embodiment, the antisense compounds of the invention differ from the native DNA by modification of the phosphodiester backbone to extend the lifetime of the antisense oligonucleotide, wherein the phosphate substituent is replaced with a phosphorothioate. Likewise, one or both ends of the oligonucleotide may be substituted with one or more acridine derivatives that intercalate between adjacent base pairs within the nucleic acid strand.

An alternative to antisense is the use of "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in mammals. The natural function of RNAi and co-suppression appears to be to protect the genome from invasion by mobile genetic elements such as retrotransposons and viruses, which when active, produce abnormal RNA or dsRNA in host cells (see, e.g., jensen, J. Et al, nat. Genet.21:209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molecule, each RNA strand having a length of about 19 to 25 (e.g., 19 23 nucleotides). For example, dsRNA molecules useful in the methods of the invention may comprise RNAs corresponding to the sequences listed in table 4 and complements thereof. Preferably, at least one strand of the RNA has a 3' overhang of 1-5 nucleotides. The synthesized RNA strands are combined under conditions that form a double-stranded molecule. The RNA sequence may comprise at least 8 nucleotide portions of SEQ ID NO. 4, which may have a total length of 25 nucleotides or less. The design of siRNA sequences for a given target is within the ordinary skill of those in the art. Commercial services are available (Qiagen, valencia, calf) that design siRNA sequences and ensure that at least 70% of expression knockdown.

The dsRNA can be administered as a pharmaceutical composition and by known methods, wherein the nucleic acid is introduced into the desired target cell. Common gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viral methods. Such methods are taught in Ausubel et al Current Protocols in Molecular Biology, john Wiley & Sons, inc.

Ribozymes may also be used to reduce the amount and/or biological activity of MASP-2, e.g., ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules that cleave nucleic acid molecules having sequences that are wholly or partially homologous to the sequence of the ribozyme. Ribozyme transgenes can be designed that encode RNA ribozymes that specifically pair with the target RNA and cleave the phosphodiester backbone at specific positions, thereby functionally inactivating the target RNA. Upon such cleavage, the ribozyme itself is not altered and is therefore capable of recycling and cleaving other molecules. The inclusion of a ribozyme sequence within the antisense RNA confers RNA cleavage activity thereto, thereby increasing the activity of the antisense construct.

Ribozymes useful in the practice of the present invention generally comprise a hybridization region of at least about nine nucleotides that is complementary in nucleotide sequence to at least a portion of a target MASP-2mRNA and a catalytic region that is suitable for cleavage of the target MASP-2mRNA (see generally EPA No. 0 321 201; WO88/04300; haseloff, J. Et al, nature 334:585-591, 1988; fedor, M.J. et al, proc.Natl.Acad.Sci.USA 87:1668-1672, 1990; cech, T.R. Et al, ann.Rev.biochem.55:599-629, 1986).

The ribozyme may be directed to the cell in the form of an RNA oligonucleotide incorporating the ribozyme sequence, or introduced into the cell as an expression vector encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same manner as described for antisense polynucleotides.

Antisense RNA and DNA, ribozymes and RNAi molecules useful in the methods of the present invention can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These include techniques well known in the art for chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences may be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as the T7 or SP6 polymerase promoters. Alternatively, depending on the promoter used, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly may be stably introduced into the cell line.

Various well-known modifications of DNA molecules can be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides at the 5' and/or 3' end of the molecule, or the use of phosphorothioates or 2' O-methyl groups instead of phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

V. pharmaceutical compositions and methods of delivery

Administration of drugs

In another aspect, the invention provides a composition for inhibiting adverse effects of MASP-2 dependent complement activation in a subject suffering from a disease or condition as disclosed herein, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. MASP-2 inhibitors may be administered to a subject in need thereof in a therapeutically effective dose to treat or ameliorate a condition associated with MASP-2 dependent complement activation. A therapeutically effective dose refers to an amount of MASP-2 inhibitor sufficient to result in an improvement in symptoms associated with the disease or condition.

Toxicity and therapeutic efficacy of MASP-2 inhibitors may be determined by standard pharmaceutical procedures employing experimental animal models, such as the murine MASP-2-/-mouse model described in Experimental example 1, which express human MASP-2 transgenes. Using such animal models, NOAEL (no adverse effect level observed) and MED (minimum effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, expressed as the ratio NOAEL/MED. MASP-2 inhibitors that exhibit large therapeutic ratios or indices are most preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in a human. The dosage of MASP-2 inhibitor is preferably within the range of circulating concentrations that include MEDs that are less toxic or nontoxic. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

In some embodiments, the therapeutic efficacy of a MASP-2 inhibitor for treating, inhibiting, reducing, or preventing fibrosis in a mammalian subject having or at risk of developing a disease or disorder caused by or exacerbated by fibrosis and/or inflammation is determined by one or more of: reduction of one or more markers of inflammation and scarring (e.g., tgfβ -1, CTFF, IL-6, apoptosis, fibronectin, laminin, collagen, EMT, infiltrating macrophages) in kidney tissue; reduction of soluble markers of inflammatory and fibrotic kidney disease released into urine and plasma (e.g., by measurement of renal excretion function).

For any compound formulation, an animal model can be used to estimate a therapeutically effective dose. For example, doses may be formulated in animal models to achieve a circulating plasma concentration range that includes MED. Quantitative levels of MASP-2 inhibitor in plasma may also be measured, for example, by high performance liquid chromatography.

In addition to toxicity studies, effective dosages may be estimated based on the amount of MASP-2 protein present in the living subject and the binding affinity of the MASP-2 inhibitor. MASP-2 levels in normal human subjects have been shown to be present in serum at low levels in the range of 500ng/ml, and MASP-2 levels in particular subjects can be determined using a quantitative assay for MASP-2, described in Moller-Kristensen M. Et al, J.Immunol. Methods 282:159-167, 2003.

Generally, the dosage of the administered composition comprising the MASP-2 inhibitor will vary depending upon such factors as the age, weight, height, sex, general medical condition and prior medical history of the subject. Illustratively, MASP-2 inhibitors, such as anti-MASP-2 antibodies, may be administered in a dosage range of about 0.010 to 10.0mg/kg, preferably 0.010 to 1.0mg/kg, more preferably 0.010 to 0.1mg/kg of subject body weight. In some embodiments, the compositions comprise a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

The therapeutic efficacy and appropriate dosage of the MASP-2 inhibitory compositions and methods of the invention in a given subject can be determined according to complement assays well known to those of skill in the art. Complement produces numerous specific products. In the last decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a and C5a, and the large activation fragments iC3b, C4d, bb and sC5b-9. Most of these assays utilize monoclonal antibodies that react with the neoantigen (neoantigen) exposed on the fragment, but not with the antigen on the native protein from which they are formed, making these assays very simple and specific. While radioimmunoassays are sometimes still used for C3a and C5a, most rely on ELISA techniques. Rear part (S) These assays measure both the unprocessed and its 'desArg' fragments, which are the predominant form found in the circulation. Unprocessed fragment and C5a _desArg Rapid clearance is obtained by binding to cell surface receptors and is therefore present in very low concentrations, whereas C3a _desArg Does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway independent indicator of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of the liquid phase product sC5b-9 of activation of the membrane attack pathway provides evidence that complement is activated to completion. Since both the lectin and classical pathways produce the same activation products C4a and C4d, measurement of both fragments does not provide any information as to which of the two pathways has produced the activation product.

Inhibition of MASP-2 dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administration of a MASP-2 inhibitor according to the methods of the invention: inhibition of the production or production of MASP-2 dependent complement activation system products C4b, C3a, C5a, and/or C5b-9 (MAC) (e.g., as measured in example 2), reduction of C4 cleavage and C4b deposition (e.g., as measured in example 10), or reduction of C3 cleavage and C3b deposition (e.g., as measured in example 10).

Additional reagents

In certain embodiments, a method of preventing, treating, reversing, and/or inhibiting fibrosis and/or inflammation comprises administering a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) as part of a therapeutic regimen, along with one or more other drugs, biologicals, or therapeutic interventions suitable for inhibiting fibrosis and/or inflammation. In certain embodiments, the additional pharmaceutical, biological, or therapeutic intervention is appropriate for the particular symptom associated with the disease or condition caused by or exacerbated by fibrosis and/or inflammation. For example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen, along with one or more immunosuppressants (e.g., methotrexate, cyclophosphamide, azathioprine, and mycophenolate). As a further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen, along with one or more agents designed to increase blood flow (e.g., nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As a further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen, along with one or more agents that are expected to reduce fibrosis (e.g., d-penicillamine, colchicine, PUVA, relaxin, cyclosporin, tgfβ blockers, or p38 MAPK blockers). As a further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen, along with a steroid or bronchodilator.

Compositions and methods comprising MASP-2 inhibitors (e.g., MASP-2 inhibitory antibodies) may optionally comprise one or more additional therapeutic agents that may potentiate the activity of the MASP-2 inhibitor or provide related therapeutic functions in a additive or synergistic manner. For example, in the context of treating a subject having a disease or condition caused by or exacerbated by fibrosis and/or inflammation, one or more MASP-2 inhibitors may be administered (including co-administration) in combination with one or more additional anti-fibrotic agents, and/or one or more antiviral and/or anti-inflammatory agents and/or immunosuppressants.

MASP-2 inhibitors (e.g., MASP-2 inhibitory antibodies) may be used in combination with other therapeutic agents such as general antiviral agents, or immunosuppressive agents such as corticosteroids, immunosuppressants or cytotoxic and/or anti-fibrotic agents.

In some embodiments of the methods described herein, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) is used as a monotherapy for treating a subject having coronavirus or influenza virus. In some embodiments of the methods described herein, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) is used in combination with other therapeutic agents, such as antiviral agents, therapeutic antibodies, corticosteroids, and/or other agents that have been shown to be effective in treating a subject with coronavirus or influenza virus. In some embodiments, the pharmaceutical composition comprises a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) and at least one additional therapeutic agent, such as an antiviral agent (e.g., rituximab), a therapeutic antibody against a target other than MASP-2, a corticosteroid, an anticoagulant such as low molecular weight heparin (e.g., enoxaparin), and an antibiotic (e.g., azithromycin).

In such combination therapies, the MASP-2 inhibitor may be formulated or administered simultaneously with, before or after: one or more other desired covd-19 therapeutic agents such as an antiviral agent (e.g., adefovir), a therapeutic antibody directed against a target other than MASP-2, a corticosteroid, or an anticoagulant. Each component of the combination therapy may be formulated in a variety of ways known in the art. For example, the MASP-2 inhibitor and the second agent of the combination therapy may be formulated together or separately. MASP-2 inhibitors and additional agents may be suitably administered to a patient with COVID-19 at one time or over a series of treatments.

Exemplary antiviral agents include, for example, darunavir (which may be used with ritonavir or cobicistat to increase daruna Wei Shuiping), faviravir, lopinavir, ritonavir, adefovir, gan Li Xiwei, ebastine, darunavir, ASC09, emtricitabine, tenofovir, umifnovir, balano Sha Wei, azlocarbadine and/or ISR-50. Exemplary therapeutic antibodies include, for example, an inhibitor of vascular growth factor (e.g., bevacizumab), a PD-1 blocking antibody (e.g., thymosin, carlizumab), a CCR5 antagonist (e.g., le Long Lishan antibody), an IL-6 receptor antagonist (e.g., sha Lilu monoclonal antibody, tolizumab), an IL-6 targeted inhibitor (e.g., stetuximab), an anti-GMCSF antibody (e.g., gimsilumab, TJM 2), a GMCSF receptor alpha blocking antibody (e.g., mo Fushan antibody), an anti-C5 antibody (e.g., eculizumab, lei Wuzhu monoclonal antibody), and/or an anti-C5 a antibody (IFX-1).

In some embodiments of the methods described herein, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody such as OMS646, or a small molecule inhibitor of MASP-2) is used in combination with an antiviral agent such as adefovir for treating a subject with COVID-19.

Other agents that may be effective for treating coronaviruses and/or influenza viruses include, for example, chloroquine/hydroxychloroquine, camostat mesylate, ruxolitinib, polyethylene glycol interferon alpha-2 b, lenalifode, ifenprodil, recombinant ACE2, APN01, brlacidin, BXT-25, BIO-11006, fingolimod, WP1122, interferon beta-1 a, nafamostat, losartan, and/or alteplase.

Drug carrier and delivery vehicle

In general, MASP-2 inhibitor compositions of the invention in combination with any other selected therapeutic agent are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and is selected so as not to adversely affect the biological activity of the MASP-2 inhibitor (and any other therapeutic agent in combination therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. patent No. 5,211,657 to Yamada. anti-MASP-2 antibodies and inhibitory peptides useful in the present invention may be formulated in formulations in solid, semi-solid, gel, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, depot agents, inhalants and injections, which allow for oral, parenteral or surgical administration. The present invention also contemplates topical application of the compositions by coating medical devices and the like.

Suitable carriers for parenteral delivery via injection, infusion, or irrigation and topical delivery include distilled water, physiological phosphate buffered saline, normal or lactated ringer's solution, dextrose solution, hank's solution, or propylene glycol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any biocompatible oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or ointment.

The carrier may also comprise a delivery vehicle to maintain (i.e., prolong, delay or regulate) the delivery of one or more agents, or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent. As non-limiting examples, such delivery vehicles may include microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include PEO: PHB: PEO copolymer and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2, and PEO/cyclodextrin complexes disclosed in U.S. patent application publication No. 2002/0019369A 1. Such hydrogels may be injected locally at the intended site of action, or subcutaneously or intramuscularly, to form a slow release depot.

For intra-articular delivery, the MASP-2 inhibitor may be carried in an injectable liquid or gel carrier as described above, an injectable slow release delivery vehicle as described above, or hyaluronic acid or a hyaluronic acid derivative.

For oral administration of the non-peptide agent, the MASP-2 inhibitor may be carried in an inert filler or diluent such as sucrose, cornstarch or cellulose.

For topical administration, the MASP-2 inhibitor may be carried in ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders, or in a gel or microcapsule delivery system via a transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers are under development, and may be suitably adapted to deliver the present invention in an aerosol, inhalant, or nebulized delivery vehicle, respectively.

For Intrathecal (IT) or Intraventricular (ICV) delivery, suitable sterile delivery systems (e.g., liquids; gels, suspensions, etc.) may be used to administer the present invention.

The compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, diluents, buffers, permeation enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

Pharmaceutical carrier for antibodies and peptides

More specifically, with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations may be administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid, such as water, oil, saline, glycerol or ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like, may be present in the composition comprising the anti-MASP-2 antibody and the inhibitory peptide. Additional components of the pharmaceutical composition include petrolatum (e.g., petrolatum of animal, vegetable or synthetic origin), such as soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are the preferred liquid carrier for injectable solutions.

anti-MASP-2 antibodies and inhibitory peptides may also be administered in the form of depot injections or implant formulations, which may be formulated in a manner that allows for sustained or pulsatile release of the active agent.

Pharmaceutically acceptable carrier for expression inhibitors

More specifically, with respect to the expression inhibitors useful in the methods of the present invention, compositions are provided that comprise an expression inhibitor as described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloidal dispersion system.

Pharmaceutical compositions comprising the expression inhibitor may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. The preparation of such compositions typically involves combining an expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates (including glucose, sucrose, or dextrins), chelating agents (e.g., EDTA, glutathione, and other stabilizers), and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents.

In some embodiments, the compositions may be prepared and formulated as emulsions, which are generally heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets (see Idson, vol. Pharmaceutical Dosage Forms, rieger and Banker (editions), marcek Dekker, inc., n.y., 1988). Examples of naturally occurring emulsifiers used in emulsion formulations include acacia, beeswax, lanolin, lecithin and phospholipids.

In one embodiment, the composition comprising the nucleic acid may be formulated as a microemulsion. As used herein, microemulsion refers to a system of water, oil, and amphiphilic compounds that is a single optically isotropic and thermodynamically stable liquid solution (see Rosoff, pharmaceutical Dosage Forms, volume 1). The methods of the invention can also use liposomes for the transfer and delivery of antisense oligonucleotides to desired sites.

Pharmaceutical compositions and formulations of expression inhibitors for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers can be used, as well as aqueous, powder or oily bases and thickeners and the like.

Mode of administration

Pharmaceutical compositions comprising MASP-2 inhibitors may be administered in a number of ways, depending on whether the local or systemic mode of administration is most appropriate for the condition to be treated. Further, the compositions of the present invention may be delivered by coating or incorporating the compositions onto or into implantable medical devices.

Systemic delivery

As used herein, the terms "systemic delivery" and "systemic administration" are intended to include, but are not limited to, oral and parenteral routes, including Intramuscular (IM), subcutaneous, intravenous (IV), intraarterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, and other routes of administration, which are effective to cause the delivered agent to be dispersed to a single or multiple sites of intended therapeutic effect. Preferred routes for systemic delivery of the present compositions include intravenous, intramuscular, subcutaneous, and inhalation. It will be appreciated that the exact systemic route of administration of the selected agent utilized in a particular composition of the invention will be determined to partially take into account the sensitivity of the agent to the metabolic conversion pathway associated with a given route of administration. For example, the peptide energy agent may be most suitably administered by a route other than orally.

MASP-2 inhibitory antibodies and polypeptides may be delivered to a subject in need thereof by any suitable means. Methods of delivery of MASP-2 antibodies and polypeptides include administration by oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (e.g., via a finely divided formulation), transdermal, nasal, vaginal, rectal or sublingual routes of administration, and may be formulated in a dosage form appropriate for each route of administration.

As a representative example, MASP-2 inhibitory antibodies and peptides may be introduced into the living body by application to body membranes capable of absorbing polypeptides, such as nasal, gastrointestinal and rectal membranes. The polypeptide is typically applied to the absorbent membrane in combination with a permeation enhancer. (see, e.g., lee, V.H.L., crit.Rev.Ther.Drug Carrier Sys.5:69, 1988; lee, V.H.L., J.controlled Release13:213, 1990; lee, V.H.L., edit, peptide and Protein Drug Delivery, marcel Dekker, new York (1991); deBoer, A.G., et al, J.controlled Release 13:241, 1990.) for example, STDHF is a synthetic derivative of fusidic acid, which is a steroid surfactant similar in structure to bile salts, and has been used as a permeation enhancer for nasal delivery. (Lee, W.A., biopharm.22, nov./Dec.1990.)

MASP-2 inhibitory antibodies and polypeptides may be introduced in combination with another molecule (e.g., a lipid) to protect the polypeptide from enzymatic degradation. For example, covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in vivo, and thus to extend half-life (Fuertges, F. Et al, J.controlled Release 11:139, 1990). A number of polymer systems for protein delivery have been reported (Bae, Y.H. et al, J.controlled Release 9:271, 1989; hori, R.et al, pharm.Res.6:813, 1989; yamakawa, I.et al, J.pharm.Sci.79:505, 1990; yoshihiro, I.et al, J.controlled Release 10:195, 1989; asano, M.et al, J.controlled Release 9:111, 1989; rosenblatt, J.et al, J.controlled Release 9:195, 1989; mao, K., J.controlled Release 12:235, 1990; takakura, Y.et al, J.Pharmi.78:117, 1989, takura, Y.et al, J.Pharmi.78:219, 1989).

Recently, liposomes have been developed with improved serum stability and circulation half-life (see, e.g., U.S. patent No. 5,741,516 to Webb). In addition, various methods of liposomes and liposome-like formulations as potential drug carriers have been reviewed (see, e.g., U.S. patent No. 5,567,434 to Szoka, U.S. patent No. 5,552,157 to Yagi, U.S. patent No. 5,565,213 to Nakamori, U.S. patent No. 5,738,868 to Shinkarenko, and U.S. patent No. 5,795,587 to Gao).

For transdermal applications, MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable ingredients (e.g., carriers and/or adjuvants). There are no limitations regarding the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration and do not degrade the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels or suspensions with or without purified collagen. MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be administered systemically at intervals determined to maintain the desired level of efficacy. For example, the composition may be administered every two weeks to four weeks or at less frequent intervals, such as by subcutaneous injection. The dosage regimen is determined by the physician taking into account various factors that may affect the combined action of the agents. These factors will include the extent of progression of the condition to be treated, the age, sex and weight of the patient, and other clinical factors. The dosage of each individual agent will vary according to: the presence and nature of the MASP-2 inhibitor, as well as any drug delivery vehicle (e.g., a slow release delivery vehicle) included in the composition. In addition, the number of doses may be adjusted to account for variations in the frequency of administration and pharmacokinetic behavior of the agent being delivered.

Local delivery

As used herein, the term "topical" encompasses drug administration in or around the site of intended localized action, and may include, for example, local delivery to the skin or other affected tissue, ocular delivery, intrathecal (IT), intraventricular (ICV), intra-articular, intra-luminal, intracranial, or intracapsular administration, placement, or irrigation. Topical administration may be preferred to enable lower doses to be administered to avoid systemic side effects and for more accurate control of timing of delivery and active agent concentration at the site of local delivery. Despite patient-to-patient variability in metabolism, blood flow, etc., topical administration provides a known concentration at the target site. Improved dose control is also provided by the direct delivery mode.

Local delivery of MASP-2 inhibitors may be accomplished in the context of a surgical procedure for treating a disease or condition caused by or exacerbated by fibrosis and/or inflammation, for example during an operation such as surgery.

Treatment regimen

In prophylactic applications, a pharmaceutical composition comprising a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a MASP-2 inhibitory small molecule compound) is administered to a subject susceptible to, or otherwise at risk of developing: the amount of the pharmaceutical composition is sufficient to inhibit MASP-2 dependent complement activation and thereby reduce, eliminate or reduce the risk of developing symptoms of respiratory syndrome. In both prophylactic and therapeutic regimens, compositions comprising a MASP-2 inhibitor may be administered in several doses until sufficient therapeutic results have been achieved in the subject. The use of the MASP-2 inhibitory compositions of the invention may be carried out by a single administration of the composition or by a defined sequence of administration for the treatment of acute conditions associated with fibrosis and/or inflammation. Alternatively, the composition may be administered at periodic intervals over an extended period of time for treating chronic conditions associated with fibrosis and/or inflammation.

In both prophylactic and therapeutic regimens, compositions comprising a MASP-2 inhibitor may be administered in several doses until sufficient therapeutic results have been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitor comprises a MASP-2 antibody, which may be suitably administered to an adult patient (e.g., 70kg average adult weight) at the following doses: 0.1mg to 10,000mg, more preferably 1.0mg to 5,000mg, still more preferably 10.0mg to 2,000mg, still more preferably 10.0mg to 1,000mg, and still more preferably 50.0mg to 500mg. For pediatric patients, the dose may be adjusted in proportion to the weight of the patient. The use of the MASP-2 inhibitory compositions of the invention may be carried out by a single administration of the composition or a defined sequence of administrations for treating a subject suffering from or at risk of developing a disease or condition caused by or exacerbated by fibrosis and/or inflammation. Alternatively, the composition may be administered at periodic intervals (e.g., daily, weekly, monthly or bi-monthly) over an extended period of time for treating a subject suffering from or at risk of developing a disease or condition caused by or exacerbated by fibrosis and/or inflammation.

In both prophylactic and therapeutic regimens, compositions comprising a MASP-2 inhibitor may be administered in several doses until sufficient therapeutic results have been achieved in the subject.

Use of MASP-2/C1-INH complex as biomarker for severe COVID-19

In another aspect, the present disclosure provides biomarkers of MASP-2-mediated lectin pathway activation, i.e., liquid phase MASP-2/C1-INH complexes, whose changes in presence and/or concentration are clinically relevant to the presence of or risk of developing an acute disease associated with a COVID-19 infection, the presence of or risk of developing one or more long-term sequelae associated with a COVID-19 infection, and/or the treatment of a COVID-19 infection with a complement inhibitor. Also provided are compositions, kits, and methods for interrogating a biological fluid, such as the concentration of liquid phase MASP-19/C1-INH complex in a biological fluid obtained from a subject infected with COVID-19. The compositions and methods are particularly useful for assessing the risk of developing an acute disease associated with covd-19, diagnosing covd-19 and/or covd-19-induced long-term sequelae, monitoring the progression or alleviation of covd-19-associated disease and/or monitoring the response to complement inhibitor (e.g., MASP-2 inhibitor) treatment or optimizing such treatment.

As described in examples 25 to 30 herein, to determine the activation state of human MASP-2 (as shown in SEQ ID NO: 6) as a Lectin Pathway (LP) effector enzyme, use is made of the feature that exploits the fact that: once MASP-2 is activated, a human C1 inhibitor (C1-INH) (shown as SEQ ID NO: 86) that acts as a pseudo-substrate forms a covalent liquid phase MASP-2/C1-INH complex. Thus, the level of MASP-2/C1-INH complex in plasma or serum samples provides a clear measure of recent LP activation.

As described in examples 25 to 30, the inventors have observed that the concentration of MASP-2/C1-INH in blood (e.g. serum and/or plasma) is abnormally high in patients with severe COVID-19 as well as in subjects previously infected with COVID-19 and suffering from long-term sequelae. The inventors have also observed that after recovery, the concentration of MASP-2/C1-INH complex is in most cases reduced to normal levels. As further described in examples 29 and 30, the inventors determined that subjects with acute covd-19 had high levels of MASP-2/C1-INH prior to treatment with the naloxone Li Shan antibody, which declined rapidly after treatment with the naloxone Li Shan antibody. The inventors believe that it is useful to monitor the increase in MASP-2/C1-INH complex concentration in patients infected with SARS-CoV-2 for: the patient is diagnosed as having or at risk of developing acute covd-19, and the subject is also diagnosed as having or at risk of developing acute post-covd-19 (also referred to as long-covd-19), and the subject identified as having such risk is optionally treated with a complement inhibitor, e.g., a MASP-2 inhibitor. Monitoring the status of MASP-2/C1-INH complex may also be used to determine whether a patient with COVID-19 is responsive to a complement inhibitor, such as MASP-2 inhibitor therapy, and optionally adjusting the dosage of MASP-2 inhibitor as needed to bring the level of MASP-2/C1-INH into the normal range.

In accordance with the foregoing, in one embodiment, the present disclosure provides, inter alia, compositions, kits and methods for measuring the amount of MASP-2/C1-INH complex as a biomarker for MASP-2-mediated lectin pathway activation, and the concentration of the MASP-2/C1-INH complex in biological fluids is abnormally elevated in patients suffering from acute COVID-19 disease associated with SARS-Cov2 infection and/or in subjects previously infected with SARS-Cov2 and suffering from or at risk of developing long-COVID-19 sequelae.

Thus, in one embodiment, the invention relates to a monoclonal antibody (mAb C#7 or mAb C#8) which specifically binds MASP-2 and is capable of binding MASP-2 complexed with C1-INH (also known as MASP-2/C1-INH complex), and the use of such an antibody in a method of detecting the presence or amount of MASP-2/C1-INH complex in a biological sample. In another embodiment, the invention relates to an immunoassay comprising the use of MASP-2 specific monoclonal antibodies and C1-INH specific antibodies to measure the presence or amount of MASP-2/C1-INH complex in a mammalian subject suffering from or at risk of developing a coronavirus or influenza virus infection to determine the activation state of the lectin pathway, optionally before and after treatment with a complement inhibitor such as a MASP-2 inhibitor, e.g., a MASP-2 inhibitory antibody (e.g., a nano-Li Shan antibody), wherein the MASP-2 inhibitory antibody is capable of inhibiting the lectin pathway.

In one embodiment, the presence or amount of MASP-2/C1-INH complex can be used as a biomarker for determining the presence or risk of developing severe or long-term COVID-19 in a subject infected with SARS-CoV-2, wherein a higher level of MASP-2/C1-INH in the subject compared to a normal uninfected subject or population or threshold of subjects indicates that the subject has severe COVID-19, or has a higher risk of developing severe COVID-19, or has long-COVID-19, or has an increased risk of developing long-COVID-19. In some embodiments, the method further comprises administering a complement inhibitor to a subject determined to have an increased level of MASP-2/C1-INH complex. In some embodiments, the present disclosure provides methods of determining the efficacy of a complement inhibitor, e.g., a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, e.g., a nascidin Li Shan antibody) in a subject and/or monitoring administration in a subject undergoing treatment with a complement inhibitor. In some embodiments, the subject has a coronavirus, such as a covd-19 or influenza virus or other lectin pathway disease or disorder (e.g., HSCT-TMA, igAN, gvHD or other lectin pathway disease or disorder).

A. Highly sensitive ELISA assays and anti-MASP-2 monoclonal antibodies in bead-based assays for detection of MASP-2/C1-INH complexes in biological samples

As described in examples 25 to 30 herein, the inventors have generated anti-MASP-2 antibody mAb clones #C7 and #C8 suitable for use in detection assays of MASP-2/C1-INH complexes and in the methods described herein.

Variable heavy and light chain fragments of mAb clone #c7 and mAb clone #c8 have been cloned and sequenced as described in examples 25 and 26.

The heavy and light chain variable regions of anti-MASP-2 mAb clones #C7 and #C8 are provided below.

SEQ ID NO. 87 mAb clone #C7HC variable region

SEQ ID NO. 88 mAb clone #C7LC variable region

SEQ ID NO. 97 mAb clone #C8HC variable region

SEQ ID NO. 98 mAb clone #C8LC variable region

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds MASP-2 that is also complexed with C1-INH, wherein the antibody comprises (a) HC-CDR1, HC-CDR2 and HC-CDR2 in the heavy chain variable region as set forth in SEQ ID NO:87 and LC-CDR1, LC-CDR2, LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR2 and HC-CDR2 in the heavy chain variable region as set forth in SEQ ID NO:97 and LC-CDR1, LC-CDR2, LC-CDR3 in the light chain variable region as set forth in SEQ ID NO: 98. In one embodiment, an isolated antibody comprises a heavy chain variable region comprising HC-CDR-1 comprising SEQ ID NO. 89, HC-CDR2 comprising SEQ ID NO. 90 and HC-CDR3 comprising SEQ ID NO. 91 and a light chain variable region comprising LC-CDR1 comprising SEQ ID NO. 92, LC-CDR2 comprising SEQ ID NO. 83 and LC-CDR3 comprising SEQ ID NO. 94. In one place In one embodiment, the anti-MASP-2 antibody is a humanized antibody, chimeric antibody or fully human antibody. In one embodiment, the anti-MASP-2 antibody fragment is selected from Fv, fab, fab ', F (ab) 2 and F (ab') ₂ . In one embodiment, the anti-MASP-2 antibody is a single chain molecule. In one embodiment, the anti-MASP-2 antibody is an IgG molecule selected from the group consisting of IgG1, igG2, and IgG 4. In one embodiment, the anti-MASP-2 antibody or antigen binding fragment thereof is labeled with a detectable moiety, such as a detectable moiety suitable for use in an immunoassay, as further described herein. In one embodiment, the anti-MASP-2 antibody or fragment thereof is immobilized on a substrate, such as a substrate suitable for use in an immunoassay, as further described herein.

In one embodiment, the anti-MASP-2 antibody or fragment thereof (i.e., an antibody or fragment thereof that specifically binds to human MASP-2 complexed with C1-INH) comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region comprising SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO:88, wherein the CDRs are numbered according to the Kabat numbering system. In one embodiment, the anti-MASP-2 antibody or fragment thereof (i.e., an antibody or fragment thereof that specifically binds to human MASP-2 complexed with C1-INH) comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region comprising SEQ ID NO:97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising the amino acid sequence SEQ ID NO. 89; (b) HC-CDR2 comprising amino acid sequence SEQ ID NO. 90, (c) HC-CDR3 comprising amino acid sequence SEQ ID NO. 91; (d) LC-CDR1 comprising the amino acid sequence SEQ ID NO. 92; (e) LC-CDR2 comprising amino acid sequence SEQ ID No. 93 and (f) LC-CDR3 comprising amino acid sequence SEQ ID No. 94.

In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH domain that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence of SEQ ID NO:87 or SEQ ID NO: 97. In one embodiment, the MASP-2-specific antibody or fragment thereof comprises a VL domain that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence of SEQ ID NO. 88. In one embodiment, the anti-MASP-2 antibody or fragment thereof contains a VH comprising SEQ ID NO 87 and a VL comprising SEQ ID NO 88 or SEQ ID NO 98.

In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising SEQ ID NO. 89, (b) HC-CDR2 comprising SEQ ID NO. 90; (c) HC-CDR3 comprising SEQ ID NO. 91; (d) LC-CDR1 comprising SEQ ID No. 92, (e) LC-CDR2 comprising SEQ ID No. 93 and (f) LC-CDR3 comprising SEQ ID No. 94. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VH domain that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence of SEQ ID NO: 87. In one embodiment, the anti-MASP-2 antibody or fragment thereof comprises a VL domain that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence of SEQ ID NO: 88. In one embodiment, the anti-MASP-2 antibody or fragment thereof contains a VH comprising SEQ ID NO. 87 and a VL comprising SEQ ID NO. 88.

In another embodiment, the present disclosure provides a nucleic acid encoding a Complementarity Determining Region (CDR) of a heavy chain variable region of an anti-MASP-2 antibody or antigen binding fragment thereof that specifically binds human MASP-2 while complexing with C1-INH, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 95, and wherein the CDRs are numbered according to the Kabat numbering system. In another embodiment, the present disclosure provides a nucleic acid encoding a Complementarity Determining Region (CDR) of a light chain variable region of an anti-MASP-2 antibody or antigen binding fragment thereof of human MASP-2 that specifically binds while complexing with C1-INH, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:96, and wherein the CDRs are numbered according to the Kabat numbering system.

In another embodiment, the present disclosure provides a cloning or expression vector comprising nucleic acid encoding a Complementarity Determining Region (CDR) of a heavy and/or light chain variable region of an antibody or antigen binding fragment that specifically binds human MASP-2, wherein (a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No. 87 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 88, or (b) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No. 97 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 98, wherein the CDRs are numbered according to the Kabat numbering system.

In another embodiment, the present disclosure provides a cell comprising a cloning or expression vector comprising a nucleic acid encoding a Complementarity Determining Region (CDR) of a heavy and/or light chain variable region of an antibody or antigen binding fragment that specifically binds human MASP-2, wherein (a) the heavy chain variable region comprises the amino acid sequence shown as SEQ ID No. 87 and the light chain variable region comprises the amino acid sequence shown as SEQ ID No. 88, or (b) wherein the heavy chain variable region comprises the amino acid sequence shown as SEQ ID No. 97 and the light chain variable region comprises the amino acid sequence shown as SEQ ID No. 98, wherein the CDRs are numbered according to the Kabat numbering system.

In another embodiment, the present disclosure provides a method for producing an anti-MASP-2 antibody comprising culturing a cell containing an expression vector containing a nucleic acid encoding one or both of the heavy and light chain polypeptides of a MASP-2 antibody or antigen binding fragment disclosed herein. The cells or cell cultures are cultured under conditions and for a time sufficient to allow the cells (or cell cultures) to express the antibody or antigen-binding fragment thereof encoded by the nucleic acid. The method may further comprise isolating the antibody or antigen-binding fragment thereof from the cell (or cell culture) or medium in which the one or more cells are cultured.

In one embodiment, the present disclosure provides compositions comprising any of the anti-MASP-2 antibodies or antigen binding fragments disclosed herein.

In one embodiment, the present disclosure provides a matrix suitable for use in an immunoassay comprising at least one or more of the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein.

In one embodiment, the present disclosure provides a kit for detecting the presence or amount of MASP-2/C1-INH complex in a test sample, such as a biological sample, comprising (a) at least one container, and (b) at least one or more of any MASP-2 antibody or antigen binding fragment disclosed herein. In some embodiments, the kit comprises an anti-MASP-2 antibody comprising a binding domain comprising (a) HC-CDR1, HC-CDR2 and HC-CDR2 in the heavy chain variable region as set forth in SEQ ID NO:87 and LC-CDR1, LC-CDR2, LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR2 and HC-CDR2 in the heavy chain variable region as set forth in SEQ ID NO:97 and LC-CDR1, LC-CDR2, LC-CDR3 in the light chain variable region as set forth in SEQ ID NO: 98. In some embodiments, the kit comprises an anti-MASP-2 antibody comprising a binding domain comprising the following six CDRs: (a) HC-CDR1 comprising SEQ ID NO. 89, (b) HC-CDR2 comprising SEQ ID NO. 90; (c) HC-CDR3 comprising SEQ ID NO. 91; (d) LC-CDR1 comprising SEQ ID No. 92, (e) LC-CDR2 comprising SEQ ID No. 93 and (f) LC-CDR3 comprising SEQ ID No. 94. In some embodiments, the kit further comprises at least one antibody that specifically binds to C1-INH.

anti-MASP-2 antibodies labeled with a detectable moiety

In another aspect, the invention provides anti-MASP-2 antibodies (e.g., mAb clone #C7 or mAb clone #C8) that are labeled with a detectable moiety (i.e., a moiety that allows detection and/or quantification). In various embodiments, the antibodies described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, antibody "conjugate" refers to an anti-MASP-2 antibody covalently linked to a detectable label. In the present invention, monoclonal antibodies, antigen-binding fragments thereof and antibody derivatives thereof, such as single chain variable fragment antibodies or epitope-tagged antibodies, may be covalently linked to a detectable label. In "direct detection", only one detectable antibody, i.e., the primary detectable antibody, is used. Thus, direct detection means that the antibody itself conjugated to the detectable label can be detected without the need to add a secondary antibody (secondary antibody).

A "detectable label" is a molecule or material that can generate a detectable (e.g., visual, electronic, or other means) signal indicative of the presence and/or concentration of the label in a sample. When conjugated to antibodies, the detectable label may be used to localize and/or quantify the target against which the specific antibody is directed. Thus, the presence and/or concentration of a target in a sample can be detected by detecting the signal generated by the detectable label. The detectable label may be detected directly or indirectly, and several different detectable labels conjugated with different specific antibodies may be used in combination to detect one or more targets.

Examples of detectable labels that can be directly detected include fluorescent dyes and radioactive materials and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after the primary antibody is applied. Thus, detection is performed by detecting the binding of the secondary antibody or binding agent to the primary detectable antibody. Examples of primary detectable binders or antibodies that require the addition of secondary binders or antibodies include enzyme detectable binders and hapten detectable binders or antibodies.

Examples of detectable labels that can be conjugated to antibodies of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5- (and 6) -carboxyfluorescein, 5-or 6-carboxyfluorescein, 6- (fluorescein) -5- (and 6) -carboxamido caproic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, cy3, and Cy5, optionally substituted coumarins including AMCA, perCP, phycobiliproteins including R-phycoerythrin (RPE) and Allophycocyanin (APC), texas red, prins red, green Fluorescent Protein (GFP) and analogs thereof, and conjugates of R-phycoerythrin or allophycocyanin, inorganic fluorescent labels such as particles based on semiconductor materials such as coated CdSe nanocrystals.

Examples of polymeric particle labels include microparticles or latex particles of polystyrene, PMMA or silica gel (which may incorporate fluorescent dyes), or polymeric micelles or capsules containing dyes, enzymes or substrates.

Examples of metal particle markers include gold particles convertible by silver staining agents and coated gold particles. Examples of haptens include DNP, fluorescein Isothiocyanate (FITC), biotin and digoxygenin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), beta-Galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, beta-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and Glucose Oxidase (GO). Examples of commonly used substrates for horseradish peroxidase include 3,3' -Diaminobenzidine (DAB), nickel-extended diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine Dihydrochloride (BDHC), hanker-Yates reagent (HYR), indofine Blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphthol-pyronine (alpha-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2- (p-iodophenyl) -3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), 5-bromo-4-chloro-3-indolyloxy-beta-D-galactoside/ferroferricyanide (BCIG/FF).

Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B1-phosphate/fast red TR (NABP/FR), naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), naphthol-AS-B1-phosphate/-fast red TR (NABP/FR), naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), naphthol-AS-B1-phosphate/new red (NABP/NF), bromochloroindolyl phosphate/nitroblue tetrazole (BCIP/NBT), 5-bromo-4-chloro-3-indolyl-B-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1, 2-dioxetane and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioisotopes of iodine, cobalt, selenium, tritium, carbon, sulfur and phosphorus.

The detectable label may be attached to an antibody described herein (i.e., any anti-MASP-2 antibody or anti-C1-INH antibody) or any other molecule that specifically binds to the biomarker of interest, such as an antibody, nucleic acid probe, or polymer. Furthermore, one of ordinary skill in the art will appreciate that the detectable label may also be conjugated to a second, and/or third, and/or fourth, and/or fifth binding agent or antibody, etc. Furthermore, the skilled person will appreciate that each additional binding agent or antibody used to characterize the biomarker of interest may serve as a signal amplification step. The biomarkers can be visually detected using, for example, an optical microscope, a fluorescence microscope, an electron microscope, wherein the detectable substance is, for example, a dye, colloidal gold particles, a luminescent reagent. Visually detectable substances that bind to the biomarker may also be detected using a spectrophotometer. Where the detectable substance is a radioisotope, it may be detected visually by autoradiography, or non-visually using a scintillation counter. See, e.g., larsson,1988,Immunocytochemistry:Theory and Practice, (CRC Press, boca Raton, fla.); methods in Molecular Biology, vol.80 1998,John D.Pound (code) (Humana Press, totowa, n.j.).

In another embodiment, the anti-MASP-2 antibody (e.g., mAb clone #C7, mAb clone #C8 or anti-C1-INH antibody) is unlabeled (i.e., naked), and its presence may be detected using a labeled antibody that binds to the anti-MASP-2 antibody or the anti-C1-INH antibody.

B. Compositions and kits for measuring MASP-2/C1-INH complexes in biological samples

Composition and method for producing the same

In another aspect, the present disclosure provides a substrate, such as a solid support (e.g., an insoluble substrate, such as a non-aqueous substrate, such as a plate or slide made of glass, polysaccharide (e.g., agarose), polyacrylamide, polystyrene, plastic, or metal, polymer-coated beads, tubes, or ceramic or metal chips), comprising immobilized (or otherwise deposited) monoclonal anti-MASP-2 antibodies disclosed herein (e.g., anti-MASP-2 antibodies that bind MASP-2 complexed with C1-INH, such as mAb clone #c7 or mAb clone #c8). In some embodiments, the anti-MASP-2 antibodies are immobilized (or deposited) at discrete locations (e.g., in wells of a multi-layer plate, or in an array on a biochip). In some embodiments, the matrix comprising anti-MASP-2 antibodies may be part of a kit for detecting MASP-2/C1-INH complexes in a biological sample obtained from a mammalian subject.

Kit for detecting a substance in a sample

In another aspect, the present disclosure provides a kit for performing one or more assays disclosed herein.

In one embodiment, the present disclosure provides a kit (i.e., a packaged combination of a predetermined amount of reagents) having reagents and instructions for detecting the presence or amount of MASP-2/C1-INH complex in a test sample, such as a biological sample. An exemplary kit may contain at least one anti-MASP-2 monoclonal antibody or antigen-binding fragment thereof (i.e., mAb clone #C7 or mAb clone #C8) as described herein and at least one anti-C1-INH antibody. In the case where the anti-MASP-2 antibody or anti-C1-INH antibody is labeled with a detectable moiety, such as an enzyme, the kit will include the substrate and cofactor required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents can be widely varied to provide concentrations in the reagent solutions, which substantially optimize the sensitivity of the assay. In particular, the reagents may be provided as a dry powder, typically lyophilized, including excipients which, when dissolved, will provide a reagent solution having the appropriate concentration.

In addition, the kit may include instructional materials (e.g., for detecting MASP-2/C1-INH complex, or the absence thereof) disclosing the manner of use of the antibodies of the invention. For example, the kit may additionally contain means for detecting the label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, a suitable secondary label such as sheep anti-mouse HRP, etc.). The kit may additionally include buffers and other reagents conventionally used to perform particular immunoassays, as is well known in the art.

Certain embodiments provide a kit for detecting the presence or amount of MASP-2/C1-INH in a sample, wherein the kit contains at least one anti-MASP-2 antibody as described herein, e.g., an antibody or fragment comprising CDRs from clone #C7 as shown in Table 5 or an antibody or fragment comprising CDRs from clone #C8. In certain embodiments, the kit may comprise buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the invention are attached, and the like, as well as instructions for use.

Certain embodiments provide a kit for detecting the presence or amount of MASP-2/C1-INH in a biological sample, wherein the kit contains at least one anti-MASP-2 antibody as described herein, e.g., an antibody or fragment comprising CDRs from MASP-2-specific clone mAb #C7 or an antibody or fragment comprising CDRs from clone #C8 as set forth in Table 5. The subject anti-MASP-2 antibodies and antigen binding fragments thereof may be labeled with any suitable detectable moiety as described herein. In certain embodiments, the kit may comprise buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the invention are attached, and the like, as well as instructions for use.

The items in the kit may be individually wrapped or packaged in separate containers, which together are provided in a larger container (e.g., a cardboard or polystyrene foam box).

In accordance with the foregoing, in one embodiment, the present disclosure provides a kit for measuring the presence or amount of MASP-2/C1-INH complex in a biological sample, the kit comprising at least one monoclonal antibody that specifically binds MASP-2 in an immunoassay and optionally an anti-C1-INH specific antibody or antigen binding fragment thereof that specifically binds C1-INH. In one embodiment, the MASP-2-specific antibody or fragment thereof comprises a binding domain comprising HC-CDR-1, HC-CDR-2 and HC-CDR-3 in the heavy chain variable region comprising SEQ ID NO. 87 and comprising LC-CDR-1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO. 88, wherein the CDRs are numbered according to the Kabat numbering system. In one embodiment, the MASP-2-specific antibody or fragment thereof comprises a binding domain comprising HC-CDR-1, HC-CDR-2 and HC-CDR-3 in the heavy chain variable region comprising SEQ ID NO. 97 and comprising LC-CDR-1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO. 98, wherein the CDRs are numbered according to the Kabat numbering system.

In some embodiments, the kit further comprises at least one container.

In some embodiments, the kit is for performing an enzyme-linked immunosorbent assay (ELISA). In one embodiment, the anti-MASP-2 antibody or fragment thereof is a coated antibody. In one embodiment, the anti-MASP-2 antibody or fragment thereof is a detection antibody. In one embodiment, the C1-INH-specific antibody or fragment thereof is a coated antibody. In one embodiment, the C1-INH-specific antibody or fragment thereof is a detection antibody. In one embodiment, the anti-MASP-2 antibody is a coated/captured antibody and comprises a binding domain comprising (a) HC-CDR-1, HC-CDR-2 and HC-CDR-3 in the heavy chain variable region comprising SEQ ID NO:87 and comprising LC-CDR-1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO:88, or (b)) HC-CDR-1, HC-CDR-2 and HC-CDR 3 in the heavy chain variable region comprising SEQ ID NO:97 and comprising LC-CDR-1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system and are immobilized on a substrate, such as a solid support (e.g., an insoluble substrate, such as a non-aqueous substrate, e.g., a plate or slide made of glass, polysaccharide (e.g., agarose), polyacrylamide, polystyrene, plastic or metal coated plates, tubes or ceramic or metal chips).

In some embodiments, the kit is for performing a bead-based immunofluorescence assay, such as a Luminex assay, and comprises (i) at least one anti-MASP-2 antibody, such as mAb #c7 or mAb #c8, immobilized on beads (e.g., polystyrene microspheres or magnetic polystyrene microspheres), which is suitable for capturing MASP-2/C1-INH complexes from human serum or plasma. In some embodiments, the kit further comprises (ii) at least one anti-C1-INH antibody, for use as a detection antibody to detect the captured complex. In one embodiment, the kit further comprises (iii) an anti-C1 s antibody suitable for capturing the C1s/C1-INH complex from human serum or plasma.

In various embodiments of the kits of the invention, the subject antibodies and antigen-binding fragments thereof may be labeled with any suitable detectable moiety as described herein. In certain embodiments, the kit further comprises buffers, enzymes, labels, substrates, beads (e.g., polystyrene or magnetic polystyrene microspheres), or other surfaces to which the antibodies of the invention are attached, and the like, as well as instructions for use.

C. Method for detecting MASP-2/C1-INH complexes in biological samples

As described herein, the inventors have generated anti-MASP-2 antibodies suitable for use in immunoassays for detecting the presence and/or amount of MASP-2/C1-INH in biological samples, such as biological samples obtained from mammalian subjects.

In one aspect, the anti-MASP-2 antibodies of the invention (e.g., mAb clone #C7 or mAb clone #C8) are used in an in vitro immunoassay for analysis of a test sample, e.g., a biological sample obtained from a test subject, for the presence or amount of MASP-2/C1-INH complex. In such in vitro immunoassays, the anti-MASP-2 antibody or antigen binding fragment thereof may be naked or may be labeled with a detectable moiety, as described herein, and may be used in the liquid phase or bound to a matrix, as described below. For the purposes of in vitro assays, any type of antibody, e.g., murine, chimeric, humanized or human antibodies, may be utilized, as the host immune response need not be considered.

Antibodies of the disclosure can be used in any known immunoassay method, such as competitive binding assays, direct and indirect sandwich assays, lateral flow assays (e.g., dipstick format), bead-based assays, and immunoprecipitation assays (see, e.g., zola, monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC press. Inc., 1987).

The sandwich assay involves the use of two antibodies, each capable of binding to a different immunogenic portion of the MASP-2/C1-INH complex to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody (e.g., an anti-MASP-2 antibody, such as clone #C7 or clone #C8, which is immobilized on a solid support (e.g., matrix), and then a second antibody binds to C1-INH, thus forming an insoluble three-part complex. The secondary antibody itself may be labeled with a detectable moiety (direct sandwich assay), or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay).

For example, one preferred type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. ELISA assays, regardless of the detection system employed, typically involve immobilization of the antigen or antibody to a substrate (e.g., a solid support), and the use of appropriate detection reagents. In ELISA assays, the protein antigen-antibody reaction occurs on a substrate (e.g., a solid support), typically in wells on a microtiter plate. The antigen and the primary antibody (also referred to as a coating or capture antibody) react and produce a stable complex that can be visualized by the addition of a secondary antibody, referred to as a detection antibody, which can be directly or indirectly linked to the enzyme. The addition of the substrate of the enzyme results in the formation of a color, which can be measured photometrically.

In one embodiment, the anti-MASP-2 antibodies of the invention (e.g., clone #C7 or clone #C8) are used as coating/capture antibodies to detect the presence of MASP-2/C1-INH complexes in biological samples using enzyme-linked immunosorbent assays (ELISA) (see, e.g., gold et al J Clin Oncol.24:252-58, 2006).

In a direct competition ELISA, a pure or semi-pure antigen preparation binds to a matrix insoluble in the fluid or cell extract being tested and a quantity of a detectably labeled soluble antibody is added to allow detection and/or quantification of the binary complex formed between the matrix-bound antigen and the labeled antibody.

In contrast, a "double-determinant" ELISA, also known as a "two-site ELISA" or "sandwich assay", requires a small amount of antigen, and the assay does not require a large amount of purified antigen. Thus, the double determinant ELISA is superior to the direct competition ELISA for detecting antigens in clinical samples. See, e.g., dual determinant ELISA for quantification of c-myc oncoprotein in biopsy samples. Field et al Oncogene 4:1463 (1989); spandidos et al, anti cancer Res.9:821 (1989). In a double-determinant ELISA, an amount of unlabeled monoclonal antibody or antibody fragment ("capture antibody") is bound to a matrix (e.g., a solid support), a test sample is contacted with the capture antibody, and an amount of detectably labeled soluble antibody (or antibody fragment) is added to allow detection and/or quantification of the ternary complex formed between the capture antibody, antigen, and labeled antibody.

In one embodiment, the capture antibody that is bound to a matrix (e.g., a solid support) is an anti-MASP-2 antibody or antigen-binding fragment thereof that binds MASP-2 complexed with Cl-INH as disclosed herein. In one embodiment, the capture antibody that is bound to a matrix (e.g., a solid support) is a MASP-2 specific antibody or antigen binding fragment thereof as disclosed herein.

Methods of performing a double determinant ELISA are well known to those skilled in the art. See, e.g., field et al Oncogene 4:1463 (1989); spandidos et al, anti cancer Res.9:821 (1989); and Moore et al Methods in Molecular Biology Vol10:273-281 (The Humana Press, inc. 1992).

In a double determinant ELISA, the soluble antibody or antibody fragment must bind to an epitope on the MASP-2/C1-INH complex that is different from the epitope recognized by the capture antibody. A double-determinant ELISA can be performed to determine whether MASP-2/C1-INH complexes are present in a test biological sample, such as a bodily fluid (e.g., blood, plasma, or serum) or a biopsy sample. Alternatively, an assay may be performed to quantify the amount of MASP-2/C1-INH complex present in a clinical sample of bodily fluid. Quantitative assays can be performed by including dilutions of MASP-2/C1-INH complex.

An in vitro immunoassay may be performed in which at least one MASP-2 specific antibody or antigen binding fragment thereof is bound to a substrate (e.g., a solid support). For example, MASP-2 specific monoclonal antibodies or fragments thereof may be attached to a polymer, such as aminodextran, to attach the monoclonal antibodies to an insoluble substrate, such as polymer coated beads, plates, tubes, or ceramic or metal chips. In one embodiment, the matrix is suitable for use in an ELISA method (e.g., a multi-well microtiter plate). In one embodiment, the matrix is a bead (e.g., polystyrene microsphere or magnetic polystyrene microsphere) for use in a bead-based immunofluorescent assay, such as the Luminex assay as described herein. In some embodiments, the present disclosure provides immunoassays for the detection of both MASP-2/C1-INH and Cls/C1-INH complexes, wherein MASP-2 specific antibodies or antigen binding fragments thereof are bound to one set of beads and C1 s-specific antibodies or antigen binding fragments thereof are bound to a second set of beads.

Other suitable in vitro assays will be apparent to those skilled in the art. The specific concentration of the detectably labeled anti-MASP-2 antibody or C1-INH specific antibody, the temperature and time of incubation, and other assay conditions may vary depending on a variety of factors, including the concentration of MASP-2/C1-INH complex in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine the operation of each assay and the optimal assay conditions by employing routine experimentation.

Assays for detection and/or measurement of MASP-2/C1-INH complexes

In accordance with the foregoing, in one aspect, the invention provides a method of determining the presence or amount of MASP-2/C1-INH in a test sample, e.g., a biological sample, comprising (a) contacting the test sample with a MASP-2 specific monoclonal antibody or antigen binding fragment thereof in an in vitro immunoassay; (b) Contacting the test sample with a C1-INH specific antibody, and (C) detecting the presence or absence of binding of said C1-INH antibody, wherein the presence of binding is indicative of the presence or amount of MASP-2/C1-INH complex in the sample.

In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof comprises a binding domain that contains HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region comprising SEQ ID NO 87 and contains additional LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO 88. In some embodiments, the MASP-2 specific antibody or fragment thereof is a monoclonal antibody comprising CDRs from MASP-2 specific clone #C7, as shown in Table 5. In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof comprises a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region comprising SEQ ID NO 97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region comprising SEQ ID NO 98.

In one embodiment, the method further comprises comparing the amount of MASP-2/C1-INH detected according to step (C) to a reference standard or control sample to determine the level of MASP-2/C1-INH in the test sample.

In one embodiment, the control sample is an individual or pooled sample of subjects suffering from a lectin pathway disease or disorder (e.g., covd-19, HSCT-TMA, igAN, gvHD, or other lectin pathway disease or disorder). In one embodiment, the control sample is an individual sample or a pooled sample of normal healthy volunteers. In one embodiment, the control sample is a baseline sample of the subject prior to treatment with a complement inhibitor (e.g., a MASP-2 inhibitor or other complement inhibitor). In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof is immobilized on a substrate. In one embodiment, the immunoassay is an ELISA assay. In one embodiment, the immunoassay is a bead-based assay, such as a Luminex assay.

In one embodiment, the MASP-2 specific antibody is labeled with a detectable moiety and step (b) comprises detecting the presence of the detectable moiety. In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof is naked (i.e., unlabeled) and the labeled antibody that binds to MASP-2 antibody is used to detect the presence or amount of antibody or fragment thereof that binds to the MASP-2/C1-INH complex. In one embodiment, the MASP-2 specific antibody or antigen binding fragment thereof is immobilized on a substrate (i.e., captured/coated) and the bound MASP-2/C1-INH complex is detected with a secondary antibody that binds C1-INH, as described herein.

In one embodiment, the test sample is a biological sample obtained from a mammalian subject. In various embodiments, the biological sample is a fluid sample selected from the group consisting of whole blood, serum, plasma, sputum, amniotic fluid, cerebrospinal fluid, cell lysate, ascites, urine, and saliva. In one embodiment, the biological sample is selected from the group consisting of blood, serum, plasma, urine, and cerebrospinal fluid. As described herein, in some embodiments, the assay methods and kits are suitable for measuring the presence and/or amount of MASP-2/C1-INH in low serum concentrations (i.e., less than 10% serum, e.g., 0.1% to 9%, e.g., 0.5% to 8%, e.g., 1% to 5%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% serum).

In one embodiment, the mammalian subject (e.g., human) is infected with SARS-CoV-2 and has or is at risk of developing severe and/or long-COVID-19.

In one embodiment, the mammalian subject (e.g., human) has been treated with a complement inhibitor, e.g., a lectin pathway complement inhibitor, e.g., a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, e.g., a nano-cord Li Shan antibody), as further described herein.

In one embodiment, the mammalian subject (e.g., human) has a lectin pathway disease or disorder (e.g., covd-19, HSCT-TMA, igAN, gvHD, or other lectin pathway disease or disorder).

As described herein, methods of detecting or measuring MASP-2/C1-INH complexes according to various embodiments of the disclosure can be used to define a pharmacodynamic endpoint or treatment threshold, or to determine whether to treat a subject with a complement inhibitor, e.g., a lectin pathway complement inhibitor, e.g., a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, e.g., a naso Li Shan antibody).

Although the details of the immunoassay may vary with the particular format employed, in one embodiment, the method of detecting MASP-2/C1-INH in a test sample comprises the step of contacting the test sample with a capture antibody that specifically binds MASP-2. MASP-2 antibodies are allowed to bind MASP-2/C1-INH in the sample under immunoreaction conditions and the presence of the bound antibodies is detected directly or indirectly with anti-C1-INH antibodies. The MASP-2 specific antibodies may be used as capture antibodies, for example in ELISA or bead-based assays for MASP-2/C1-INH, or as secondary antibodies that bind MASP-2/C1-INH captured by capture antibodies that bind Cl-INH. The presence of the secondary antibody is then typically detected, as is known in the art. In some embodiments, the immunoassay is performed on a solid support. In some embodiments, the immunoassay is an ELISA assay. In some embodiments, the immunoassay is a bead-based assay.

D. Methods of diagnosing, monitoring and treating subjects having or at risk of developing acute covd-19 or having or at risk of developing long-covd-19

The anti-MASP-2 antibodies, methods, reagents and kits of the invention may be used in a variety of applications. For example, in certain embodiments, the assays of the invention may be used to evaluate the level of MASP-2/C1-INH in a subject infected with SARS-CoV-2 to determine the risk of developing acute COVID-19 (i.e., acute respiratory distress syndrome, pneumonia, or some other pulmonary or other acute manifestations of COVID-19, such as thrombosis), or the likelihood of recovering from acute COVID-19 and/or the long-term sequelae associated with developing long-COVID-19 (i.e., selected from cardiovascular complications, neurological complications, kidney injury, pulmonary complications, inflammatory conditions such as Kawasaki disease, multiple system inflammatory syndrome in children, multiple system organ failure, extreme fatigue, muscle weakness, low fever, inattention, memory errors, mood changes, sleep difficulties, needle pain in the legs, diarrhea and vomiting, taste and smell, sore and difficulty in the throat, diabetes and hypertension, new episodes of respiratory depression, skin pain, palpitations, and the presence of an inhibitor of the respiratory system, e.g., human complement factor, and/or the level of the inhibitor of the human complement factor, e.g., MASP-62, in a subject, and/or the level of the inhibition of the anti-lectin pathway (e.g., MASP-2) are evaluated and/or the level of the inhibitor of the anti-1-MASP-pathway, e.g., the level of the antibodies.

In some embodiments, the assays of the invention can be used to evaluate the extent to which complement pathway inhibitors (e.g., MASP-2 inhibitors) reduce lectin complement pathway activation in vivo. In some embodiments, the methods of the invention are performed on biological samples obtained from subjects infected with SARS-CoV-2. In some embodiments, the level of MASP-2/C1-INH complex detected in the assays of the invention is compared to an appropriate reference value. The reference value may be, for example, a value measured from a sample obtained from a healthy patient (or population of healthy patients), or a value measured from a sample or a set of samples obtained from a subject suffering from severe covd-19, or a value measured from a sample obtained from a patient suffering from covd-19 undergoing treatment with a MASP-2 inhibitor (e.g., obtained prior to treatment or at a point in time in a series of treatments), or the reference value may be from healthy serum that has been activated with an agent that activates the lectin pathway (see example 25), or the reference value may be a predetermined threshold. In one embodiment, the control sample is an individual or pooled sample of subjects with acute covd-19. In one embodiment, the control sample is an individual or pooled sample of normal healthy volunteers. In one embodiment, the control sample is a baseline sample of the subject prior to treatment with a complement inhibitor (e.g., a MASP-2 inhibitor or other complement inhibitor). As described herein, methods of detecting MASP-2/C1-INH complexes according to various embodiments of the present disclosure can be used to assess the extent of complement activation of the lectin pathway and thereby to define the pharmacodynamic endpoint or therapeutic threshold of complement inhibitors or to decide whether to treat with complement inhibitors, e.g., lectin pathway complement inhibitors, e.g., MASP-2 inhibitors (e.g., MASP-2 inhibitory antibodies, e.g., nano-cord Li Shan antibodies).

E. Method for evaluating the extent of lectin pathway complement activation in mammalian subjects

In one aspect, the present disclosure provides methods of assessing the extent of lectin pathway complement (APC) activation in a test sample and performing an immunoassay comprising capturing and detecting a MASP-2/C1-INH complex in the test sample, wherein the level of MASP-2/C1-INH complex detected in the test sample is indicative of the extent of lectin pathway complement activation in the test sample. In one embodiment, the test sample is a biological sample obtained from a mammalian subject, and the method comprises the steps of: (a) Providing a biological sample obtained from a mammalian subject; and (b) assessing the extent of lectin pathway activation in a subject by performing an immunoassay according to the methods of the invention described herein, said immunoassay comprising at least one of capturing MASP-2/C1-INH complex in a biological sample and detecting the level thereof. For example, in one embodiment, the immunoassay comprises capturing and detecting MASP-2/C1-INH complexes in a test sample, wherein the MASP-2/C1-INH complexes are captured or detected with MASP-2 specific monoclonal antibodies. In various embodiments, the methods comprise comparing the level of MASP-2/C1-INH complex detected in a test sample (e.g., a biological sample) to a predetermined level or control sample, wherein the level of MASP-2/C1-INH complex detected in the test sample is indicative of the extent of lectin pathway complement activation in the test sample (e.g., the biological sample). In some embodiments, the method further comprises using the results of the comparative analysis to provide diagnostic, prognostic, or treatment-related information about the mammalian subject from which the biological sample was obtained. In some embodiments, the test sample is obtained from a subject currently infected with SARS-CoV-2, and the method is used to assess the risk of the subject developing an acute covd-19 disease, wherein an increase in the level of MASP-2/C1-INH by at least 20%, such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 2-fold, or at least 3-fold, or more, as compared to a normal healthy control (e.g., a healthy and uninfected SARS-CoV-2 subject or population of subjects) or a reference standard, is indicative of an increased risk of developing an acute covd-19 disease and/or a likelihood of recovery in a subject with an acute covd-19 disease.

In some embodiments, the test sample is obtained from a subject that has been infected with SARS-CoV-2, and the method is used to assess the risk of the subject developing a long-COVID-19 disease, wherein an increase in the level of MASP-2/C1-INH by at least 2-fold or more as compared to a normal healthy control is indicative of an increased risk of developing a long-COVID-19 disease.

In some embodiments, the disclosure provides methods of evaluating the effect of an inhibitor of human complement on lectin pathway complement activation in vivo. Any compound that binds to or otherwise blocks the production and/or activity of any complement component may be utilized in accordance with the present disclosure. For example, the complement inhibitor may be, for example, a small molecule, a nucleic acid or nucleic acid analog, a peptidomimetic, or a macromolecule that is not a nucleic acid or protein, such as an antibody or fragment thereof. In some embodiments, the disclosure provides methods of evaluating the effect of an inhibitor (e.g., an antibody or small molecule) specific for a human complement component, e.g., an inhibitor of a complement component selected from the group consisting of C1 (C1 q, C1r, C1 s), C2, C3, C4, C5, C6, C7, C8, C9, factor D, factor B, factor P, MBL, MASP-1, MASP-2, and MASP-3, on activation of the alternative complement pathway in vivo. In some embodiments, the present disclosure provides methods of evaluating the effect of an alternative complement pathway inhibitor on alternative pathway complement activation. In some embodiments, the present disclosure provides methods of evaluating the effect of a MASP-2 inhibitor on lectin pathway complement activation.

In some embodiments, the present disclosure provides methods of evaluating the effect of a MASP-2 inhibitor on lectin pathway complement activation in vivo that has been administered to a mammalian subject. In various embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody or a small molecule inhibitor of MASP-2) is administered to a mammalian subject, and a biological sample is subsequently obtained. The extent of Lectin Pathway Complement (LPC) activation in a biological sample is then assessed by performing an immunoassay according to the methods of the invention described herein, which comprises capturing and detecting MASP-2/C1-INH complex in a biological sample.

F. Methods of monitoring efficacy of MASP-2 inhibitors in mammalian subjects

In one embodiment, the present disclosure provides a method for monitoring the efficacy of treatment with a MASP-2 inhibitor in a mammalian subject, the method comprising the steps of: (a) Administering a dose of a MASP-2 inhibitor (i.e., an antibody or small molecule) to a mammalian subject at a first time point; (b) Assessing a first concentration of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (a); (c) Treating the subject with the MASP-2 inhibitory antibody at a second time point; (d) Assessing a second concentration of MASP-2/C1-INH complex in the biological sample obtained from the subject after step (C); and (e) comparing the level of MASP-2/C1-INH complex assessed in step (b) with the level of MASP-2/C1-INH complex assessed in step (d) to determine the efficacy of a MASP-2 inhibitor (i.e., an antibody or small molecule) in the mammalian subject. In one embodiment, the extent of lectin pathway activation in a subject is assessed in an immunoassay, wherein the immunoassay comprises capturing and detecting the level of MASP-2/C1-INH complex in a biological sample. Optionally, the level of MASP-2/C1-INH complex detected in the biological sample is compared to an appropriate reference value. The reference value may be, for example, a value of the MASP-2/C1-INH complex measured from a biological sample obtained from a subject prior to administration of a MASP-2 inhibitory antibody, an average value measured from samples obtained from a group of healthy control subjects, a value representing a desired degree of lectin pathway activation (e.g., a level of MASP-2/C1-INH corresponds to 90% inhibition of lectin pathway activation, or 80% inhibition, or 70% inhibition, or 60% inhibition, or 50% inhibition of lectin pathway activation). For example, a first biological sample is obtained from the subject prior to administration of the MASP-2 inhibitory antibody and a second biological sample is obtained after administration of the MASP-2 inhibitory antibody and the level of MASP-2/C1-INH complex is measured in the sample. If the level of MASP-2/C1-INH complex in the second biological sample is lower than the level of MASP-2/C1-INH complex in the first biological sample or is lower than a control value (e.g., a threshold corresponding to a percentage of inhibition of lectin pathway activation), it can be concluded that the MASP-2 inhibitory antibody inhibits lectin pathway activation to a desired extent. Alternatively, if the level of MASP-2/C1-INH complex in the second biological sample is higher than the level of MASP-2/C1-INH complex in the first biological sample, or is higher than a control value (e.g., a threshold corresponding to a percentage of inhibition of lectin pathway activation), it can be concluded that the dose of MASP-2 inhibitory antibody (e.g., nano-cord Li Shan antibody) should be increased, and optionally the method further comprises administering to the subject an increased dose of MASP-2 inhibitory antibody (e.g., nano-cord Li Shan antibody). In some embodiments, if an increased dose of MASP-2 inhibitory antibody is administered to the subject, steps (b) through (e) are repeated to determine if the increased dose of MASP-2 inhibitory antibody compared to the respective control or reference standard is sufficient to adjust the level of MASP-2/C1-INH complex to the desired level.

In some embodiments, the methods are used to monitor the efficacy of a MASP-2 inhibitory antibody that is administered to a human subject suffering from or at risk of developing a lectin pathway disease or disorder, e.g., wherein the lectin pathway disease or disorder is selected from an acute COVID-19 disease, long-COVID-19, or other lectin pathway disease or disorder (e.g., HSCT-TMA, igAN, gvHD or other lectin pathway disease or disorder).

G. Methods of diagnosing, monitoring and treating subjects with or at risk of developing severe or long-covd-19

In one embodiment, the present disclosure provides a method of determining the presence or amount of MASP-2/C1-INH complex in a test sample of biological fluid obtained from a subject currently infected with SARS-CoV-2 or likely to be infected with SARS-CoV-2 or having severe COVID-19 or previously infected with SARS-CoV-2, the method comprising: (a) Contacting a test sample of biological fluid with an antibody that binds to human MASP-2 complexed with C1-INH in an in vitro immunoassay; and (b) detecting the presence or absence or amount of an antibody or fragment thereof that binds to the MASP-2/C1-INH complex with an antibody that binds to C1-INH, wherein detection of the presence and/or amount of MASP-2/C1-INH indicates MASP-2 mediated lectin pathway activation in the subject.

In another embodiment, the present disclosure provides a method of evaluating the extent of MASP-2 mediated complement activation of the lectin pathway in a test sample of biological fluid from a subject known to be infected with SARS-CoV-2, likely to be infected with SARS-CoV-2, having severe COVID-19 or previously infected with SARS-CoV-2, comprising: (a) Providing a test sample of biological fluid obtained from a subject known to be infected with SARS-CoV-2, who is likely to be infected with SARS-CoV-2, who has severe COVID-19 or who has been previously infected with SARS-CoV-2; (b) Performing an immunoassay comprising capturing and detecting a MASP-2/C1-INH complex in a test sample, and (C) comparing the presence and/or amount of MASP-2/C1-INH detected in the test sample to a reference standard, wherein the presence or increased amount of MASP-2/CI-INH compared to the reference sample indicates an increase in MASP-2 mediated complement lectin pathway in the subject, which indicates that (i) the subject is presently suffering from a MASP-2 mediated COVID-19 disease and may benefit from treatment with a complement inhibitor, e.g., a MASP-2 inhibitor (anti-MASP-2 antibody, e.g., a nano-Li Shan antibody or a small molecule inhibitor of MASP-2), or (iii) the subject is at increased risk of developing a COVID-19-related complication, or (iii) the subject is previously infected with COVID-19 and is suffering from one or more long-term sequelae.g., is in the long-term sequelae.g., in the development of COVID-19, and is at increased risk of acute mortality, e.g., in the subject. In some embodiments, the methods further comprise administering to a subject having an increased amount of MASP-2/C1-INH complex a therapeutic agent, such as a complement inhibitor, such as a MASP-2 inhibitory antibody or a small molecule, such as a Nasog Li Shan antibody, for treating COVID-19. In some embodiments, a subject infected with COVID-19 exhibits symptoms of COVID-19. In some embodiments, the subject infected with covd-19 is asymptomatic. In some embodiments, the subject was previously infected with covd-19 and has or is at risk of developing one or more long-term sequelae associated with covd-19. In some embodiments, the method further comprises determining the level of C1s/C1-INH complex in the test sample, wherein an increase in the level of C1s/C1-INH complex (i.e., at least 2-fold or more) as compared to a healthy control indicates an increased likelihood of recovery from covd-19, and a lower level of C1s/C1-INH indicates an increased likelihood of adverse outcome.

In another aspect, the present disclosure provides a method for monitoring the efficacy of treatment with a MASP-2 inhibitory antibody in a mammalian subject having one or more complications associated with COVID-19, the method comprising: (a) Administering a dose of MASP-2 inhibitory antibody to a mammalian subject at a first time point; (b) Assessing a first concentration of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (a); (c) Treating the subject with the MASP-2 inhibitory antibody at a second time point; (d) Assessing a second concentration of MASP-2/C1-INH complex in the biological sample obtained from the subject after step (C); and (e) comparing the level of MASP-2/C1-INH complex assessed in step (b) with the level of MASP-2/C1-INH complex assessed in step (d) to determine the efficacy of the MASP-2 inhibitory antibody in the mammalian subject.

In another aspect, the present disclosure provides a method of treating a mammalian subject having or at risk of developing a covd-19 related disease or disorder, comprising if the subject is determined to have: (i) Administering to a subject an amount of MASP-2/C1-INH complex in one or more biological samples taken from the subject that is higher than a predetermined MASP-2/C1-INH complex level or than a MASP-2/C1-INH complex level in one or more control samples.

Exemplary embodiments VII

A. MASP-2 specific mAbs that bind MASP-2 complexed with C1-INH (MASP-2/C1-INH complex)

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 complexed with C1-INH, wherein the antibody comprises a binding domain comprising (a) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:98,

wherein the CDRs are numbered according to the Kabat numbering system.

2. The monoclonal antibody of paragraph 1, wherein the antibody comprises (a) a heavy chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 87 and a light chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 88 or (b) a heavy chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 97 and a light chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO. 98.

3. The monoclonal antibody of paragraph 1, wherein the antibody is a humanized antibody, a chimeric antibody or a fully human antibody.

4. The monoclonal antibody or fragment thereof of any one of paragraphs 1 to 3, wherein the antibody fragment is selected from the group consisting of Fv, fab, fab ', F (ab) 2 and F (ab') 2.

5. The monoclonal antibody of any one of paragraphs 1 to 4, wherein the antibody is a single chain molecule.

6. The monoclonal antibody of any one of paragraphs 1 to 4, wherein the antibody is an IgG molecule selected from the group consisting of IgG1, igG2 and IgG 4.

7. The monoclonal antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 6, wherein the antibody or antigen-binding fragment thereof has a K of less than 10nM _D Binds human MASP-2.

8. The monoclonal antibody, or antigen-binding fragment thereof, of any one of paragraphs 1 to 7, wherein the antibody is labeled with a detectable moiety.

9. The monoclonal antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 8, wherein the antibody or fragment thereof is immobilized on a substrate.

10. A nucleic acid molecule encoding an amino acid sequence of an antibody or fragment thereof as described in any one of paragraphs 1-7 that specifically binds human MASP-2.

11. An expression cassette comprising a nucleic acid molecule according to paragraph 10 that encodes an antibody or fragment thereof that specifically binds human MASP-2 of the invention.

12. A cell comprising at least one of the nucleic acid molecules according to paragraph 10 or paragraph 11 encoding an antibody or fragment thereof that specifically binds human MASP-2 of the invention.

13. A composition comprising an antibody or fragment thereof that specifically binds human MASP-2 as described in any one of paragraphs 1 to 9.

14. A matrix for use in an immunoassay comprising at least one antibody or fragment thereof as described in any one of paragraphs 1 to 9 that specifically binds human MASP-2.

15. A kit for detecting the presence or amount of MASP-2/C1-INH complex in a test sample, said kit comprising (a) at least one container, and (b) at least one antibody or fragment thereof as described in any one of paragraphs 1 to 9 that specifically binds human MASP-2.

16. The kit of paragraph 15, further comprising at least one antibody or fragment thereof that specifically detects C1-INH complexed with MASP-2.

17. The kit of paragraphs 15 or 16, wherein the antibody that specifically binds MASP-2 is immobilized on a substrate (e.g., a bead).

18. The kit of paragraph 16, wherein the antibody that specifically binds to C1-INH is labeled with a detectable moiety.

19. The kit of any one of paragraphs 15-18, wherein the kit is for use in an immunoassay.

20. The kit of paragraph 19, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a bead-based assay.

21. The kit of paragraphs 19 or 20, wherein the antibody or fragment thereof that binds MASP-2 is a coated/captured antibody.

22. The kit of paragraph 19 or 20, wherein the antibody or fragment thereof that binds to C1-INH is a detection antibody.

23. The kit of any of paragraphs 15-22, wherein the kit further comprises a reference standard corresponding to MASP-2/C1-INH complex levels in a healthy control subject or population of healthy human subjects.

24. The kit of any one of paragraphs 15-23, wherein the kit further comprises a reference standard corresponding to the level of MASP-2/C1-INH complex in a subject with severe COVID-19 or a population of subjects with severe COVID-19, or an amount of recombinant MASP-2/C1-INH complex corresponding to a subject with severe COVID-19.

25. The kit of any one of paragraphs 15-24, wherein the kit further comprises an antibody or fragment thereof that binds to C1s that is concurrently complexed with C1-INH.

26. The kit of paragraph 25, wherein the antibody that binds Cis is a capture antibody.

27. The kit of paragraph 25 or 26, wherein the antibody that specifically binds to C1s-INH is immobilized on a substrate (e.g., a bead).

B. Method for detecting the amount of MASP-2/C1-INH complex in a biological sample

1. A method of measuring the amount of MASP-2/C1-INH in a biological sample comprising:

(a) Providing a test biological sample from a human subject;

(b) Performing an immunoassay comprising capturing and detecting MASP-2/C1-INH complexes in a test sample, wherein MASP-2/C1-INH is captured with a monoclonal antibody that specifically binds to human MASP-2; and detecting MASP-2/C1-INH complex directly or indirectly with an antibody that specifically binds to C1-INH; and

(c) Comparing the level of MASP-2/C1-INH complex detected according to (b) with a predetermined level or a control sample, wherein the level of MASP-2/C1-INH complex detected in the test sample is indicative of the extent of lectin pathway complement activation.

2. The method of paragraph 1, wherein the biological sample is a fluid sample selected from the group consisting of whole blood, serum, plasma, urine, and cerebrospinal fluid.

3. The method of paragraph 1 or 2, wherein the antibody that specifically binds MASP-2 comprises a binding domain comprising (a) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as shown in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as shown in SEQ ID NO:97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as shown in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

4. The method of paragraph 1 or 2, wherein the biological sample is a serum sample having a concentration of 0.3% to 5%.

5. The method of any one of paragraphs 1 to 4, wherein the human subject is currently infected with SARS-CoV-2, or has been previously infected with SARS-CoV-2, or wherein the subject has or is at risk of developing another lectin pathway disease or disorder (e.g., COVID-19, HSCT-TMA, igAN, gvHD).

6. The method of paragraph 5, wherein the method further comprises determining that the subject has or is at risk of developing a severe covd-19 disease or long-covd-19 based on determining that the level of detected MASP-2/C1-INH complex is higher (at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 2-fold higher) than a predetermined level or a control reference from a healthy subject.

7. The method of any one of paragraphs 1 to 6, wherein the subject is determined to have a higher than normal level of MASP-2/C1-INH complex and is identified as a candidate for treatment with a complement inhibitor.

8. The method of any of paragraphs 1-7, wherein the method further comprises administering a complement inhibitor to a subject identified as having a higher than normal level of MASP-2/C1-INH complex.

9. The method of paragraph 8, wherein the complement inhibitor is a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, such as a nano-cable Li Shan antibody or a small molecule inhibitor of MASP-2).

10. The method of any of paragraphs 1 to 4, wherein the mammalian subject has been treated with a complement inhibitor, e.g., a lectin complement pathway inhibitor, e.g., a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, e.g., a nano-cord Li Shan antibody).

11. The method of paragraph 10, wherein the control sample is a sample taken from the subject prior to treatment with the MASP-2 inhibitor or at an earlier point in time during the course of treatment with the MASP-2 inhibitor.

12. The method of any of paragraphs 10 or 11, wherein the MASP-2 inhibitor is a MASP-2 inhibitory antibody.

C. Method for determining risk of a subject infected with SARS-CoV-2 to develop a COVID-19-associated ARDS or other adverse outcome due to acute COVID-19 or long-term sequelae associated with COVID-19

1. A method of determining the risk of a subject infected or already infected with SARS-CoV-2 to develop a covd-19-associated ARDS or other adverse consequences caused by acute covd-19 or long-term sequelae associated with covd-19, comprising:

(a) Obtaining a biological sample from the subject;

(b) Measuring the level of MASP-2/C1-INH complex in the sample;

(c) Comparing the measured level with a predetermined level of MASP-2/C1-INH complex or a reference standard to assess the risk of developing ARDS associated with COVID-19 or other adverse consequences caused by acute COVID-19 and/or long-term sequelae associated with COVID-19; and

(d) Determining the risk of the subject developing a covd-19 related ARDS or other adverse outcome caused by acute covd-19 and/or long-term sequelae associated with covd-19 and reporting the outcome to a patient, physician or database;

(e) Optionally, the treatment is administered to a subject determined to be likely to develop an acute disease caused by acute covd-19 and/or other adverse outcome and/or long-term sequelae associated with covd-19 infection.

2. The method of paragraph 1, wherein the level of MASP-2/C1-INH complex is measured in an immunoassay.

3. The method of paragraph 3, wherein the method comprises performing an immunoassay to measure the level of MASP-2/C1-INH complex in the biological sample.

4. The method of paragraph 2 or paragraph 3, wherein the immunoassay is an ELISA assay or a bead-based assay.

5. The method of paragraph 4, wherein the immunoassay comprises the use of a capture antibody that specifically binds MASP-2, the capture antibody comprising a binding domain comprising (a) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:87 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:88, or (b) HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

6. The method of any one of paragraphs 1-5, wherein the method further comprises assaying or otherwise determining the level of the C1s/C1-INH complex in a biological sample obtained from the subject.

D. Methods for treating, inhibiting, reducing or preventing acute covd-19 in a mammalian subject infected with SARS-CoV-2 and at risk of developing acute covd-19

1. Methods for treating, inhibiting, reducing or preventing acute respiratory distress syndrome, pneumonia or some other pulmonary or other acute manifestations of covd-19, such as thrombosis, in a mammalian subject infected with SARS-CoV-2 and at risk of developing acute covd-19 comprising

(i) Determining the level of MASP-2/C1-INH complex in a biological sample obtained from the subject, wherein an increased level of MASP-2/C1-INH complex as compared to a healthy control sample indicates an increased risk of developing one or more acute manifestations of covd-19; and

(ii) Administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation to a subject having an increased level of the MASP-2/C1-INH complex.

2. The method of paragraph 1, wherein the MASP-2 inhibitor is a MASP-2 antibody or fragment thereof.

3. The method of paragraph 2, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO. 6.

4. The method of paragraph 2, wherein the MASP-2 antibody or fragment thereof specifically binds to a polypeptide comprising SEQ ID NO. 6 with an affinity that is at least 10-fold greater than it binds to a different antigen in the complement system.

5. The method of paragraph 2, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

6. The method of paragraph 1, wherein the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1 q-dependent complement activation.

7. The method of paragraph 1, wherein the MASP-2 inhibitor is a small molecule MASP-2 inhibitory compound.

8. The method of paragraph 1, wherein the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

9. The method of paragraph 2, wherein the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence as set forth in SEQ ID NO. 67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence as set forth in SEQ ID NO. 69.

10. The method of paragraph 2, wherein the MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO. 67 and a light chain variable region comprising SEQ ID NO. 69.

11. The method of paragraph 1, wherein step (i) comprises using an antibody, kit or composition according to any one of paragraphs A1 to a 24.

12. The method of paragraph 1, wherein step (i) comprises the method according to any of paragraphs B1-B11.

13. The method of paragraph 1, wherein step (i) comprises the method according to any of paragraphs C1-C5.

E. Methods for treating, inhibiting, reducing or preventing long-covd-19 in a mammalian subject that has been infected with SARS-CoV-2 and is at risk of developing long-covd-19

1. A method for treating, reducing, preventing or reducing the risk of developing one or more of the long-term sequelae associated with COVID-19 in a mammalian subject having been infected with SARS-CoV-2, comprising

(i) Determining the level of MASP-2/C1-INH complex in a biological sample obtained from the subject, wherein an increased level of MASP-2/C1-INH complex as compared to a healthy control sample is indicative of an increased risk of developing one or more of the covd-19-associated long-term sequelae; and

6. The method of paragraph 2, wherein the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1 q-dependent complement activation.

11. The method of paragraph 1, wherein the one or more covd-19 associated long-term sequelae is selected from the group consisting of cardiovascular complications (including myocardial injury, cardiomyopathy, myocarditis, intravascular coagulation, stroke, venous and arterial complications, and pulmonary embolism); neurological complications (including cognitive difficulties, confusion, memory loss also known as "brain fog", headache, stroke, dizziness, syncope, seizures, anorexia, insomnia, olfactory loss, gustatory loss, myoclonus, neuropathic pain, myalgia, neurological diseases such as Alzheimer's disease, geobatwo's syndrome, miller-Fisher syndrome, the development of Parkinson's disease, kidney damage (such as Acute Kidney Injury (AKI)), pulmonary complications (including pulmonary fibrosis, dyspnea, pulmonary embolism), inflammatory conditions such as Kawasaki disease, kawasaki-like disease, multiple system inflammatory syndrome in children, and multiple system organ failure, extreme fatigue, muscle weakness, low fever, inattention, memory errors, mood changes, sleep difficulties, needle pain in arms and legs, diarrhea and vomiting, loss of taste and smell, sore throat and dysphagia, new episodes of diabetes and hypertension, rash, shortness of breath, chest pain and palpitations.

12. The method of paragraph 1, wherein step (i) comprises using an antibody, kit or composition according to any one of paragraphs A1 to a 24.

13. The method of paragraph 1, wherein step (i) comprises the method according to any of paragraphs B1-B11.

14. The method of paragraph 1, wherein step (i) comprises the method according to any of paragraphs C1-C5.

F. A method of monitoring the efficacy of treatment with a MASP-2 inhibitory antibody or antigen-binding fragment thereof in a mammalian subject in need thereof.

1. A method for monitoring the efficacy of treatment with a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, in a mammalian subject in need thereof, the method comprising:

(a) Administering a dose of a MASP-2 inhibitory antibody or antigen-binding fragment thereof to a mammalian subject at a first time point;

(b) Assessing a first level of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (a);

(c) Treating the subject with the MASP-2 inhibitory antibody or antigen-binding fragment thereof at a second time point;

(d) Assessing a second level of MASP-2/C1-INH complex in a biological sample obtained from the subject after step (C); and

(e) Comparing the level of MASP-2/C1-INH complex assessed in step (b) with the level of MASP-2/C1-INH complex assessed in step (d) to determine the efficacy of a MASP-2 inhibitory antibody or antigen binding fragment thereof in said mammalian subject.

2. The method of paragraph 1, wherein the method further comprises adjusting the dose of the MASP-2 inhibitory antibody or antigen binding fragment thereof.

3. The method of paragraph 2, wherein the dose of MASP-2 inhibitory antibody or antigen binding fragment thereof administered to the subject is increased if the level of MASP-2/C1-INH complex is higher than a control or reference standard.

4. The method of paragraph 3, wherein if an increased dose of MASP-2 inhibitory antibody or antigen binding fragment thereof is administered to the subject, steps (b) through (e) are repeated to determine if the increased dose is sufficient to adjust the level of MASP-2/C1-INH complex to the desired level as compared to the respective control or reference standard.

5. The method of paragraph 1, wherein steps (b) and (d) comprise assessing the concentration of MASP-2/CI-INH complex in the biological sample in an immunoassay.

6. The method of paragraph 5, wherein the immunoassay is a bead-based immunofluorescent assay.

7. The method of paragraph 6, wherein the immunoassay comprises the use of a capture antibody that specifically binds MASP-2, the capture antibody comprising a binding domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO. 97 and comprising LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO. 98, wherein the CDRs are numbered according to the Kabat numbering system.

8. The method of paragraph 6 or 7, wherein the biological sample is serum or plasma.

9. The method of paragraph 8, wherein the biological sample is 1% to 5% serum or plasma.

10. The method of any one of paragraphs 1-9, wherein the mammalian subject is a human subject.

11. The method of paragraph 10, wherein the human subject has or is at risk of developing a lectin pathway disease or disorder selected from the group consisting of HSCT-TMA, igAN, lupus nephritis, and graft versus host disease or some other lectin pathway disease or disorder.

12. The method of paragraph 1, wherein the human subject has or is at risk of developing COVID-19 or a long-term sequelae associated with COVID-19.

13. The method of paragraph 1, wherein the second time point is 2 to 14 days after the first time point.

14. The method of paragraph 1, wherein the second time point is within 2 to 7 days from the first time point.

15. The method of paragraph 1, wherein the second time point is within 2 to 4 days from the first time point.

VI. Examples

The following examples merely illustrate the best mode presently contemplated for practicing the invention and are not to be construed as limiting the invention. All references cited herein are expressly incorporated by reference.

Example 1

This example describes the generation of a full mouse strain with MASP-2 (MASP-2-/-) deficiency, but MAp19 (MAp19+/+) in the present example.

Materials and methods: the targeting vector pKO-NTKV 1901 was designed to disrupt the three exons encoding the C-terminus of murine MASP-2, including the exon encoding the serine protease domain, as shown in fig. 3. PKO-NTKV 1901 was used to transfect the murine ES cell line E14.1a (SV 129 Ola). Neomycin-resistant and thymidine kinase-sensitive clones were selected. 600 ES clones were screened and among these four different clones were identified and verified by southern blotting to contain predicted selective targeting and recombination events, as shown in FIG. 3. Chimeras were generated from these four positive clones by embryo transfer. The chimeras were then backcrossed in a genetic background C57/BL6 to generate transgenic males. Transgenic males were crossed with females to generate F1, with 50% of the offspring showing heterozygosity with respect to the disrupted MASP-2 gene. Heterozygous mice were mated to generate homozygous MASP-2 deficient offspring, resulting in heterozygous and wild-type mice at a 1:2:1 ratio, respectively.

Results and phenotypes: the resulting homozygous MASP-2-/-deficient homozygous mice were found to be alive and fertile and verified as MASP-2 deficient by southern blotting to confirm the correct targeting event, the absence of MASP-2mRNA by northern blotting and the absence of MASP-2 protein by western blotting (data not shown). The presence of MAp19mRNA and the absence of MASP-2mRNA was further confirmed on the LightCycler machine using time resolved RT-PCR. MASP-2-/-mice did continue to express MAP19, MASP-1 and MASP-3mRNA and protein as predicted (data not shown). The presence and abundance of the mRNA for properdin, factor B, factor D, C, C2 and C3 in MASP-2-/-mice was assessed by the LightCycler assay and found to be equivalent to the wild-type littermate control (data not shown). As further described in example 2, plasma from homozygous MASP-2-/-mice is completely devoid of lectin pathway-mediated complement activation.

MASP-2-/-lines were generated on a pure C57BL6 background: MASP-2-/-mice were backcrossed to the pure C57BL6 line for 9 passages before the MASP-2-/-line was used as an experimental animal model.

Transgenic mouse lines were also generated that were murine MASP-2-/-, MAp19++, and expressed human MASP-2 transgenes (murine MASP-2 knockout and human MASP-2 knock-in) as follows:

materials and methods: the minigene encoding human MASP-2, shown in FIG. 4, designated "mini hMASP-2" (SEQ ID NO: 49), was constructed, which includes the promoter region of the human MASP 2 gene, including the first 3 exons (exon 1 through exon 3), followed by a cDNA sequence representing the coding sequence of the next 8 exons, thus encoding the full length MASP-2 protein driven by its endogenous promoter. The mini hMASP-2 construct is injected into the fertilized egg of MASP-2-/-to replace the defective murine MASP 2 gene by human MASP-2 expressed by the transgene.

Example 2

This example demonstrates that MASP-2 is required to activate complement via the lectin pathway.

Methods and materials:

lectin pathway specific C4 cleavage assay: the C4 cleavage assay has been described by Petersen et al, J.Immunol. Methods 257:107 (2001), which measures lectin pathway activation due to lipoteichoic acid (LTA) from Staphylococcus aureus (S.aureus), which binds L-fiber gellin. The assay described by Petersen et al, (2001) is suitable for measuring lectin pathway activation via MBL by coating the plates with LPS and mannan or zymosan prior to addition of serum from MASP-2-/-mice, as described below. The assay was also modified to eliminate the possibility of C4 cleavage due to the classical pathway. This is achieved by diluting the buffer with a sample containing 1M NaCl, which allows high affinity binding of lectin pathway recognition components to their ligands, but prevents activation of endogenous C4, thereby excluding the classical pathway from participation by dissociating the C1 complex. Briefly, in a modified assay, a serum sample (diluted in high salt (1M NaCl) buffer) is added to a ligand-coated plate, followed by the addition of a constant amount of purified C4 in buffer with physiological concentration of salt. The binding recognition complex containing MASP-2 cleaves C4, resulting in C4b deposition.

The measuring method comprises the following steps:

1) Nunc Maxisorb microtitration plateNunc, catalog number 442404,Fisher Scientific) in coating buffer (15 mM Na ₂ CO ₃ 、35mM NaHCO ₃ 1. Mu.g/ml mannose diluted in pH 9.6)Glycans (M7504 Sigma) or any other ligand (e.g., the ligands listed below).

The following reagents were used in the assay:

a. mannans (1 μg/well of mannans in 100 μl of coating buffer (M7504 Sigma)):

b. zymosan (1. Mu.g/Kong Jiaomu glycan (Sigma) in 100. Mu.l coating buffer);

LTA (1. Mu.g/well in 100. Mu.l coating buffer or 2. Mu.g/well in 20. Mu.l methanol)

d. 1 mu g H-fiber-gel-specific Mab 4H5 in coating buffer

e. PSA from balloon Green (Aerococcus viridans) (2. Mu.g/well in 100. Mu.l coating buffer)

f. 100 μl/well of formalin-fixed staphylococcus aureus DSM20233 (od550=0.5) in coating buffer.

2) The plates were incubated overnight at 4 ℃.

3) After overnight incubation, the plates were blocked with 0.1% HAS-TBS buffer (in 10mM Tris-CL, 140mM NaCl, 1.5mM NaN ₃ Incubation with 0.1% (w/v) HSA in pH 7.4 for 1-3 hours followed by TBS/tween/Ca ²⁺ (with 0.05% Tween 20 and 5mM CaCl) ₂ 、1mM MgCl ₂ pH 7.4) plates were washed 3X to saturate the residual protein binding sites.

4) Serum samples to be tested were diluted in MBL binding buffer (1M NaCl) and the diluted samples were added to the plate and incubated overnight at 4 ℃. Wells that received buffer only served as negative controls.

5) After overnight incubation at 4 ℃, plates were incubated with TBS/tween/Ca ²⁺ Wash 3X. Human C4 (100. Mu.l/well in BBS (4 mM barbital, 145mM NaCl, 2mM CaCl) ₂ 、1mM MgCl ₂ pH 7.4) was added to the plate and incubated at 37℃for 90 minutes. The plates were reused with TBS/tween/Ca ²⁺ Wash 3X.

6) C4b deposition chicken anti-human C4C conjugated with alkaline phosphatase (in TBS/tween/Ca ²⁺ Diluted 1:1000) of the sample, and then the sample is subjected to detectionThe chicken anti-human C4C was added to the plates and incubated for 90 minutes at room temperature. The plates were then reused with TBS/tween/Ca ²⁺ Wash 3X.

7) Alkaline phosphatase was detected by adding 100 μl of p-nitrophenyl phosphate substrate solution, incubating for 20 minutes at room temperature, and reading OD405 in a microtiter plate reader.

Results: FIGS. 5A-B show the amount of C4B deposition on mannans (FIG. 5A) and zymosan (FIG. 5B) in serum dilutions from MASP-2+/+ (cross), MASP-2+/- (filled circles) and MASP-2+/- (filled triangles). Figure 5C shows the relative C4 convertase activity on zymosan (white bars) or mannan (shaded bars) coated plates from MASP-2/+ mice (n=5) and MASP-2-/-mice (n=4) relative to wild-type mice based on measuring the amount of C4b deposition normalized to wild-type serum. Error bars represent standard deviation. As shown in FIGS. 5A-C, plasma from MASP-2-/-mice was completely devoid of lectin pathway-mediated complement activation on mannan-and zymosan-coated plates. These results clearly demonstrate that MASP-2 is an effector component of the lectin pathway.

Recombinant MASP-2 reconstitution of lectin pathway dependent C4 activation in serum from MASP-2-/-mice

To determine that the absence of MASP-2 is a direct cause of the loss of lectin pathway dependent C4 activation in MASP-2-/-mice, the effect of adding recombinant MASP-2 protein to serum samples was examined in the C4 cleavage assay described above. Functionally active murine MASP-2 as well as catalytically inactive murine MASP-2A (wherein the active site serine residue in the serine protease domain is substituted for an alanine residue) recombinant proteins are produced and purified as described in example 3 below. Pooled serum from 4 MASP-2-/-mice was pre-incubated with recombinant murine MASP-2 or inactive recombinant murine MASP-2A at increased protein concentration and the C4 convertase activity was determined as described above.

Results: as shown in FIG. 6, the addition of functionally active murine recombinant MASP-2 protein (shown as open triangles) to serum from MASP-2-/-mice restored lectin pathway-dependent C4 activation in a protein concentration-dependent manner, whereas catalytically inactive murine MASP-2A protein (shown as star shape) did not restore C4 activation. The results shown in fig. 6 were normalized to the C4 activation observed with pooled wild-type mouse serum (shown as dashed line).

Example 3

This example describes recombinant expression and protein production of recombinant full-length human, rat and murine MASP-2, MASP-2-derived polypeptides, and catalytically inactive mutant forms of MASP-2.

Expression of full-length human, murine and rat MASP-2:

the full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI-Neo (Promega) which, under the control of the CMV enhancer/promoter region, driven eukaryotic expression (described in Kaufman R.J. et al, nucleic Acids Research 19:4485-90, 1991;Kaufman,Methods in Enzymology,185:537-66 (1991). Each of the full-length mouse cDNA (SEQ ID NO: 50) and the rat MASP-2cDNA (SEQ ID NO: 53) was subcloned into the pED expression vector. MASP-2 expression vectors were then transfected into adherent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection procedure described by Maniatis et al, 1989. Cells transfected with these constructs grew very slowly, suggesting that the encoded protease is cytotoxic.

In another method, a minigene construct (SEQ ID NO: 49) containing human cDNA for MASP-2 driven by its endogenous promoter is transiently transfected into Chinese hamster ovary Cells (CHO). Human MASP-2 protein is secreted into the medium and isolated as described below.

Full length catalytically inactive expression of MASP-2:

principle of: MASP-2 is activated by autocatalytic cleavage after recognition of the binding of the subfraction MBL or fibrous gelator (L-fibrous gelator, H-fibrous gelator or M-fibrous gelator) to its respective carbohydrate pattern. Autocatalytic cleavage leading to activation of MASP-2 often occurs during isolation procedures of MASP-2 from serum, or during purification following recombinant expression. To obtain a more stable protein preparation for use as antigen, the protein preparation is prepared by replacing serine residues present in the catalytic triad of the protease domain with rat (SEQ ID NO:55Ser617 to Ala 617); mice (SEQ ID NO:52Ser617 through Ala 617); or an alanine residue in humans (SEQ ID NO:6Ser618 to Ala 618) to produce MASP-2, which is designed as a catalytically inactive form of MASP-2A.

To generate catalytically inactive human and murine MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides shown in Table 5. The oligonucleotides in table 5 were designed to anneal to human and murine cDNA regions encoding enzymatically active serine and contained mismatches to change the serine codon to an alanine codon. For example, PCR oligonucleotides SEQ ID NO:56-59 were used in combination with human MASP-2cDNA (SEQ ID NO: 4) to amplify the region from the start codon to the enzymatically active serine and from serine to the stop codon to generate the complete open reading frame of mutant MASP-2A containing the Ser618 to Ala618 mutation. The PCR products were purified after agarose gel electrophoresis and band preparation and single adenosine overlap was generated using standard tailing procedures. The adenosine-tailed MASP-2A was then cloned into pGEM-T easy vector and transformed into E.coli.

The catalytically inactive rat MASP-2A protein was produced by combining these two oligonucleotides in equimolar amounts, heating at 100℃for 2 minutes and slowly cooling to room temperature, and kinase (kinase) and annealing SEQ ID NO. 64 and SEQ ID NO. 65. The resulting annealed fragment had Pst1 and Xba1 compatible ends, and was inserted in place of the Pst1-Xba1 fragment of wild-type rat MASP-2cDNA (SEQ ID NO: 53) to generate rat MASP-2A.

Human, murine and rat MASP-2A were each further subcloned into mammalian expression vectors pED or pCI-Neo and transfected into the Chinese hamster ovary cell line DXB1 as described below.

In another approach, the catalytically inactive form of MASP-2 is constructed using the method described in Chen et al, J.biol.chem.,276 (28): 25894-25902, 2001. Briefly, plasmids containing full-length human MASP-2cDNA (described in Thiel et al, nature 386:506, 1997) were digested with Xho1 and EcoR1, and MASP-2cDNA (described herein as SEQ ID NO: 4) was cloned into the corresponding restriction sites of the pFastBac1 baculovirus transfer vector (Life Technologies, NY). Then, the MASP-2 serine protease active site at Ser618 was changed to Ala618 by substituting the double-stranded oligonucleotides encoding the peptide region amino acids 610-625 (SEQ ID NO: 13) with the natural region amino acids 610-625 to produce a MASP-2 full-length polypeptide with an inactive protease domain.

Construction of expression plasmids containing polypeptide regions derived from human MASP-2

MASP-2 signal peptide (residues 1-15 of SEQ ID NO: 5) was used to generate the following constructs to secrete the various domains of MASP-2. Constructs expressing the human MASP-2CUBI domain (SEQ ID NO: 8) were prepared by PCR amplification of the region encoding residues 1-121 of MASP-2 (SEQ ID NO: 6) corresponding to the N-terminal CUBI domain. Constructs expressing the human MASP-2CUBIEGF domain (SEQ ID NO: 9) were prepared by PCR amplification of the region encoding residues 1-166 of MASP-2 (SEQ ID NO: 6) corresponding to the N-terminal CUBIEGF domain. A construct expressing the human MASP-2CUBIEGFCUBII domain (SEQ ID NO: 10) was prepared by PCR amplification of the region encoding residues 1-293 of MASP-2 (SEQ ID NO: 6) corresponding to the N-terminal CUBIEGFCUBII domain. According to the determined PCR method, vent was used _R The above-mentioned domains were amplified by PCR using the polymerase and pBS-MASP-2 as templates. 5 'primer sequence of sense primer (5' -CG)GGATCCATGAGGCTGCTGACCCTC-3'SEQ ID NO: 34) a BamHI restriction site (underlined) was introduced at the 5' end of the PCR product. The antisense primers shown in Table 5 below for each MASP-2 domain were designed to introduce a stop codon (bold) at the end of each PCR product, followed by an EcoRI site (underlined). Once amplified, the DNA fragment was digested with BamHI and EcoRI and cloned into the corresponding sites of the pFastBac1 vector. The resulting constructs were characterized by restriction mapping and confirmed by dsDNA sequencing.

Table 5: MASP-2PCR primer

Recombinant eukaryotic expression of MASP-2 and protein production of enzymatically inactive mouse, rat and human MASP-2A

The MASP-2 and MASP-2A expression constructs described above were transfected into DXB1 cells using standard calcium phosphate transfection procedures (Maniatis et al, 1989). MASP-2A was produced in serum-free medium to ensure that the formulation was not contaminated with other serum proteins. Media was harvested from the fused cells every other day (four total). The level of recombinant MASP-2A averaged approximately 1.5mg/L of medium for each of these three species.

MASP-2A protein purification: MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A agarose columns. This strategy makes possible rapid purification without the use of foreign tags. MASP-2A (100-200 ml was loaded with equal volumes of loading buffer (50mM Tris Cl,pH 7.5, containing 150mM NaCl and 25mM CaCl) ₂ ) Diluted medium), loaded onto MBP agarose affinity column (4 ml) pre-equilibrated with 10ml loading buffer. After washing with a further 10ml loading buffer, the protein was eluted in a 1ml fraction with 50mM Tris Cl,pH 7.5 containing 1.25M NaCl and 10mM EDTA. MASP-2A containing fractions were identified by SDS-polyacrylamide gel electrophoresis. MASP-2A was further purified by ion exchange chromatography on a MonoQ column (HR 5/5), if necessary. The proteins were dialyzed against 50mM Tris-Cl pH 7.5 containing 50mM NaCl and then loaded onto columns equilibrated in the same buffer. After washing, the bound MASP-2A was eluted with a gradient of 0.05-1M NaCl exceeding 10 ml.

Results: yields of 0.25-0.5mg MASP-2A protein were obtained from 200ml of medium. Due to glycosylation, the 77.5kDa molecular weight as determined by MALDI-MS is greater than the calculated for the unmodified polypeptide (73.5 kDa). Glycan attachment at each of the N-glycosylation sites accounts for the observed mass. MASP-2A migrated as a single band on SDS-polyacrylamide gel, confirming that it was not proteolytically processed during biosynthesis. The weight average molecular weight determined by equilibrium ultracentrifugation is consistent with the calculated value of homodimers of glycosylated polypeptides.

Production of recombinant human MASP-2 polypeptides

Another method for producing recombinant MASP-2 and MASP-2A-derived polypeptides is described in Thielens, N.M. et al, J.Immunol.166:5068-5077, 2001. Briefly, spodoptera frugiperda (Spodoptera frugiperda) insect cells (Ready-Plaque Sf9 cells obtained from Novagen, madison, WI) were grown and maintained in Sf900II serum-free medium (Life Technologies) supplemented with 50IU/ml penicillin and 50mg/ml streptomycin (Life Technologies). Trichoplusia ni (High Five) insect cells (supplied by Jadwiga Chroboczek, institut de Biologie Structurale, grenobele, france) were maintained in TC100 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, brumath, france) supplemented with 50IU/ml penicillin and 50mg/ml streptomycin. Recombinant baculoviruses use Bac-to-Bac (Life Technologies). The bacmid DNA was purified using the Qiagen midi prep purification system (Qiagen) and cellfectin in Sf900 II SFM medium (Life Technologies) was used for transfection of Sf9 insect cells as described in the manufacturer's protocol. Recombinant viral particles were collected after 4 days, titrated by viral plaque assay, and amplified as described by King and Possee, the Baculovirus Expression System: A Laboratory Guide, chapman and Hall Ltd., london, pages 111-114, 1992.

High Five cells (1.75 x 10) were infected with a recombinant virus containing MASP-2 polypeptide at a multiplicity of 2 in Sf900 II SFM medium at 28 ℃ ⁷ Individual cells/175-cm ² Tissue culture flasks) for 96 hours. By centrifugation and collectionThe supernatant was collected and diisopropyl fluorophosphate was added to a final concentration of 1 mM.

MASP-2 polypeptide is secreted into the culture medium. Culture supernatant was directed to 50mM NaCl, 1mM CaCl ₂ 50mM triethanolamine hydrochloride, pH 8.1, and loaded onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8X 12 cm) equilibrated in the same buffer at 1.5 ml/min. Elution was performed by applying a linear gradient of 1.2 liters to 350mM NaCl in the same buffer. Fractions containing recombinant MASP-2 polypeptide were identified by Western blot analysis by addition of (NH ₄ ) ₂ SO ₄ Precipitation was performed to 60% (w/v) and allowed to stand overnight at 4 ℃. The pellet was resuspended in 145mM NaCl, 1mM CaCl ₂ 50mM triethanolamine hydrochloride, pH 7.4, and applied to a TSK G3000 SWG column (7.5 x 600 mM) (Tosohaas, montgomeryville, pa.) equilibrated in the same buffer. The purified polypeptide was then concentrated to 0.3mg/ml by ultrafiltration on a Microsep microconcentrator (m.w. cutoff = 10,000) (Filtron, karlstein, germany).

Example 4

This example describes methods for producing polyclonal antibodies to MASP-2 polypeptides.

Materials and methods:

MASP-2 antigen: polyclonal anti-human MASP-2 antisera were generated by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 isolated from serum (SEQ ID NO: 6); recombinant human MASP-2 (SEQ ID NO: 6), MASP-2A comprising an inactive protease domain (SEQ ID NO: 13) as described in example 3; and recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ ID NO: 9) and CUBEGFCUBII (SEQ ID NO: 10) expressed as described in example 3 above.

Polyclonal antibodies: six week old rabbits primed with BCG (BCG vaccine) were immunized by injection in sterile saline solution with 100. Mu.g/ml of 100. Mu.g MASP-2 polypeptide. Injection was completed every 4 weeks, with antibody titers monitored by ELISA assay as described in example 5. The culture supernatant was collected for purification of the antibodies by protein a affinity chromatography.

Example 5

This example describes a method for producing murine monoclonal antibodies to a rat or human MASP-2 polypeptide.

Materials and methods:

male A/J mice (Harlan, houston, tex.) of 8-12 weeks old were subcutaneously injected with 100. Mu.g of human or rat rMASP-2 and rMASP-2A polypeptides (prepared as described in example 3) in 200. Mu.l Phosphate Buffered Saline (PBS) complete Freund's adjuvant (Difco Laboratories, detroit, mich.) pH 7.4. At two week intervals, mice were injected subcutaneously with 50. Mu.g of human or rat rMASP-2 or rMASP-2A polypeptide in incomplete Freund's adjuvant. At the fourth week, mice were injected with 50. Mu.g of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and fused 4 days later.

For each fusion, a single cell suspension was prepared from the spleen of the immunized mice and used for fusion with Sp2/0 myeloma cells. 5x108 Sp2/0 cells and 5x108 spleen cells were fused in medium containing 50% polyethylene glycol (M.W.1450) (Kodak, rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma Chemical Co., st. Louis, mo.). The cells were then adjusted to a concentration of 1.5x105 splenocytes per 200 μl of suspension in Iscove's medium (Gibco, grand Island, N.Y.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. 200 microliters of the cell suspension was added to each well of about twenty 96-well microplates. After about ten days, culture supernatants were withdrawn for screening for reactivity with purified factor MASP-2 in ELISA assays.

ELISA assay: purified hMASP-2 or rat rMASP-2 (or rMASP-2A) by adding 50. Mu.l of 50ng/ml at room temperatureWells of the 2 (Dynatech Laboratories, chantilly, va.) microplate were coated overnight. The low concentration of MASP-2 used for coating enables the selection of antibodies with high affinity. After removal of the coating solution by a flick plate, 200. Mu.l of a PBS solution of BLOTTO (skim milk powder) was addedOne hour in each well to block non-specific sites. After one hour, the wells were then washed with buffer PBST (PBS containing 0.05% Tween 20). 50 microliters of culture supernatant from each fusion well was collected and mixed with 50 microliters of BLOTTO and then added to each well of the microplate. After one hour of incubation, wells were washed with PBST. Bound murine antibodies were then detected by reaction with horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearch Laboratories, west Grove, pa.) and diluted 1:2,000 in BLOTTO. A peroxidase substrate solution containing 0.1%3, 5 tetramethylbenzidine (Sigma, st. Louis, mo.) and 0.0003% hydrogen peroxide (Sigma) was added to the wells for 30 minutes of color development. The reaction was stopped by adding 50. Mu.l of 2M H2SO 4/well. Use- >ELISA Reader(Instruments, winioski, vt.) and the optical density of the reaction mixture at 450nm was read.

MASP-2 binding assay:

culture supernatants that are positive for the MASP-2ELISA assay described above may be tested in a binding assay to determine the binding affinity of MASP-2 inhibitors for MASP-2. Similar assays can also be used to determine whether an inhibitor binds to other antigens in the complement system.

Polystyrene microtiter plate wells (96 well medium binding plates, corning Costar, cambridge, mass.) were coated overnight at 4℃with MASP-2 (20 ng/100 μl/well, advanced Research Technology, san Diego, calif.) in Phosphate Buffered Saline (PBS) pH 7.4. After aspiration of the MASP-2 solution, the wells were blocked with PBS containing 1% bovine serum albumin (BSA; sigma Chemical) for 2 hours at room temperature. Wells without MASP-2 coating served as background controls. Hybridoma supernatants or aliquots of purified anti-MASP-2 MoAb at different concentrations in blocking solution were added to the wells. After 2 hours of incubation at room temperature, wells were washed thoroughly with PBS. MASP-2 binding anti-MASP-2 MoAb was detected by adding peroxidase conjugated goat anti-mouse IgG (Sigma Chemical) to the blocking solution, which was allowed to incubate for 1 hour at room temperature. The plate was again rinsed thoroughly with PBS and 100. Mu.l of 3,3', 5' -Tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, gaithersburg, md.) was added. The reaction of TMB was quenched by the addition of 100 μl of 1M phosphoric acid and the plate was read at 450nm in a microplate reader (SPECTRA MAX 250,Molecular Devices,Sunnyvale,CA).

Culture supernatants from positive wells were then tested for their ability to inhibit complement activation in a functional assay such as the C4 cleavage assay described in example 2. Cells in the positive wells were then cloned by limiting dilution. The reactivity of the MoAb with hMASP-2 was again tested in ELISA assays as described above. Selected hybridomas were grown in spin flasks and spent culture supernatants were collected for antibody purification by protein a affinity chromatography.

Example 6

This example describes the generation and production of humanized murine anti-MASP-2 antibodies and antibody fragments.

Murine anti-MASP-2 monoclonal antibodies were generated in male A/J mice as described in example 5. The murine antibodies are then humanized to reduce their immunogenicity by replacing the murine constant regions with their human counterparts to generate chimeric IgG and Fab fragments of antibodies, as described below, which can be used in accordance with the invention to inhibit the adverse effects of MASP-2 dependent complement activation in human subjects.

1. The anti-MASP-2 variable region gene was cloned from murine hybridoma cells. Total RNA was isolated from anti-MASP-2 MoAb secreting hybridoma cells (obtained as described in example 7) using RNAzol following the manufacturer's protocol (Biotech, houston, tex.). First strand cDNA was synthesized from total RNA using oligo dT as a primer. PCR was performed using 3 'primers derived from the immunoglobulin constant C region, and degenerate primer sets derived from the leader peptide or first framework region of the murine VH or VK gene as 5' primers. Anchor PCR was performed as described by Chen and Platuscas (Chen, P.F., scand.J.Immunol.35:539-549, 1992). To clone the VK gene, a double stranded cDNA was prepared using Notl-MAK1 primer (5 '-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO: 38). Annealed adaptors AD1 (5 '-GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO: 39) and AD2 (5 '-TCCGAGAATAACGAGTG-3' SEQ ID NO: 40) were ligated to both the 5 'and 3' ends of the double stranded cDNA. The adaptors at the 3' end were removed by NotI digestion. The digested product was then used as template in PCR with AD1 oligonucleotide as 5 'primer and MAK2 (5' -CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3'SEQ ID NO: 41) as 3' primer. A DNA fragment of about 500bp was cloned into pUC 19. Several clones were selected for sequence analysis to verify that the cloned sequences contained predicted murine immunoglobulin constant regions. Not1-MAK1 and MAK2 oligonucleotides are derived from the VK region and are 182 and 84bp downstream from the first base pair of the Cκ gene, respectively. Clones comprising intact VK and leader peptide were selected.

To clone the VH gene, a double stranded cDNA was prepared using the Not1 MAG1 primer (5 '-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO: 42). Annealed adaptors AD1 and AD2 are ligated to both the 5 'and 3' ends of the double-stranded cDNA. The adaptors at the 3' end were removed by NotI digestion. The digested product was used as template in PCR with AD1 oligonucleotide and MAG2 (5 '-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. A DNA fragment of 500 to 600bp in length was cloned into pUC 19. Not1-MAG1 and MAG2 oligonucleotides were derived from the murine C.gamma.7.1 region and were 180 and 93bp downstream from the first bp of the murine C.gamma.7.1 gene, respectively. Clones containing intact VH and leader peptide were selected.

2. Construction of expression vectors for chimeric MASP-2 IgG and Fab. The cloned VH and VK genes described above were used as templates in PCR reactions to add Kozak consensus sequences to the 5 'end of the nucleotide sequences and splice donors to the 3' end of the nucleotide sequences. After sequence analysis to confirm the absence of PCR errors, VH and VK were inserted into expression vector cassettes containing human c.γ1 and c.κ, respectively, to yield pSV2neoVH-hucγ1 and pSV2neoV-hucγ. CsCl gradient purified plasmid DNA of heavy and light chain vectors was used to transfect COS cells by electroporation. After 48 hours, the culture supernatants were tested by ELISA to confirm the presence of approximately 200ng/ml chimeric IgG. Cells were harvested and total RNA was prepared. First strand cDNA was synthesized from total RNA using oligo dT as a primer. The cDNA was used as a template in PCR to generate Fd and κ DNA fragments. For the Fd gene, PCR was performed using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ ID NO: 44) as the 5 'primer and CH 1-derived 3' primer (5 '-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO: 45). The DNA sequence was confirmed to contain the complete VH and CH1 domains of human IgG 1. After digestion with the appropriate enzymes, the Fd DNA fragment was inserted at the HindIII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from various sources: pBR322 DNA (thin line) contains a DNA replication origin (pBR ori) of pBR322 and a lactamase ampicillin resistance gene (Amp); SV40 DNA represented and marked by wider hatching contains the DNA replication origin of SV40 (SV 40 ori), the early promoter (5 'of dhfr and neo genes) and the polyadenylation signal (3' of dhfr and neo genes). The SV 40-derived polyadenylation signal (pA) is also placed at the 3' end of the Fd gene.

For the kappa gene, PCR was performed using 5'-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO: 46) as a 5 'primer and a CK-derived 3' primer (5 '-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 47). The DNA sequence was confirmed to contain the complete VK and human CK regions. After digestion with the appropriate restriction enzymes, the kappa DNA fragment was inserted at the HindIII and BamHI restriction sites of the expression vector pSV2neo-TUS to give pSV2neoK. Expression of both Fd and kappa genes is driven by HCMV-derived enhancer and promoter elements. Since the Fd gene does not include cysteine amino acid residues that are involved in interchain disulfide bonds, such recombinant chimeric Fab contains non-covalently linked heavy and light chains. Such chimeric fabs are designated as cfabs.

To obtain a recombinant Fab with disulfide bonds between the heavy and light chains, the Fd gene described above can be extended to include a coding sequence of 9 additional amino acids from the hinge region of human IgG1 (EPKSCDKTH SEQ ID NO: 48). The BstEII-BamHI DNA segment encoding 30 amino acids at the 3' end of the Fd gene can be replaced with a DNA segment encoding extended Fd, resulting in pSV2dhfrFd/9aa.

3. Expression and purification of chimeric anti-MASP-2 IgG

To generate chimeric anti-MASP-2 IgG secreting cell lines, NSO cells were transfected with purified plasmid DNA of pSV2neoVH-huC. Gamma.1 and pSV2neoV-huC kappa by electroporation. Transfected cells were selected in the presence of 0.7mg/ml G418. Cells were grown in 250ml spin flasks using serum-containing medium.

100ml of the culture supernatant of the rotary culture was loaded onto a 10-ml PROSEP-A column (Bioprocessing, inc., princeton, N.J.). The column was washed with 10 bed volumes of PBS. Bound antibody was eluted with 50mM citrate buffer, pH 3.0. An equal volume of 1m hepes, pH 8.0, was added to the purified antibody containing fraction to adjust the pH to 7.0. Residual salts were removed by ultrafiltration through a Millipore membrane (m.w. cut-off: 3,000) by buffer exchange with PBS. The protein concentration of the purified antibodies was determined by BCA method (Pierce).

4. Expression and purification of chimeric anti-MASP-2 Fab

To generate a chimeric anti-MASP-2 Fab secreting cell line, CHO cells were transfected by electroporation with purified plasmid DNA of pSV2dhfrFd (or pSV2dhfrFd/9 aa) and pSV2neoκ. Transfected cells were selected in the presence of G418 and methotrexate. The selected cell lines were expanded at increasing concentrations of methotrexate. Cells were subcloned by limiting dilution into single cells. The highly productive monoclonal cell lines were then grown in 100ml of rotating culture using serum-free medium.

Chimeric anti-MASP-2 Fab was purified by affinity chromatography using a mouse anti-idiotype MoAb against MASP-2 MoAb. Anti-idiotype MASP-2 MoAb can be prepared by immunizing mice with murine anti-MASP-2 MoAb conjugated to Keyhole Limpet Hemocyanin (KLH) and screening for specific MoAb binding that can compete with human MASP-2. For purification, 100ml of supernatant from the rotating cultures of cFab or cFab/9aa producing CHO cells was loaded onto an affinity column coupled to anti-idiotype MASP-2 MoAb. The column was then washed thoroughly with PBS and the bound Fab was eluted with 50mM diethylamine, pH 11.5. Residual salts were removed by buffer exchange as described above. The protein concentration of purified Fab was determined by BCA method (Pierce).

The ability of chimeric MASP-2 IgG, cFab and cFAb/9aa to inhibit the MASP-2 dependent complement pathway can be determined by using the inhibition assay described in example 2 or example 7.

Example 7

This example describes an in vitro C4 cleavage assay for use as a functional screen to identify MASP-2 inhibitors capable of blocking MASP-2 dependent complement activation via L-fiber gel protein/P35, H-fiber gel protein, M-fiber gel protein or mannans.

C4 cleavage assay: the C4 cleavage assay has been described by Petersen, S.V. et al, J.Immunol. Methods 257:107, 2001, which measures lectin pathway activation due to lipoteichoic acid (LTA) from Staphylococcus aureus, which binds L-fiber gelling protein.

Reagent: formalin-fixed staphylococcus aureus (DSM 20233) was prepared as follows: bacteria were grown overnight at 37 ℃ in tryptic soy blood medium, washed 3 times with PBS, then fixed in PBS/0.5% formalin for 1 hour at room temperature, then further washed 3 times with PBS, then resuspended in coating buffer (15 mM Na ₂ Co ₃ 、35mM NaHCO ₃ pH 9.6).

And (3) measuring: nuncWells of microtiter plates (Nalgene Nunc International, rochester, NY) were coated with: 100 μl of formalin fixed staphylococcus aureus DSM20233 (od550=0.5) in coating buffer with 1 μ g L-fiber gel protein in coating buffer. After overnight incubation, wells were blocked with 0.1% Human Serum Albumin (HSA) in TBS (10 mM Tris HCl, 140mM NaCl,pH 7.4) followed by a solution containing 0.05% Tween 20 and 5mM CaCl ₂ Is washed with TBS (washing buffer). Human serum samples were run in 20mM Tris-HCl, 1M NaCl, 10mM CaCl ₂ Dilutions were made in 0.05% triton X-100, 0.1% HSA, pH 7.4, which prevented endogenous C4 activation and dissociated the C1 complex (consisting of C1q, C1r and C1 s). MASP-2 include anti-MASP-2 MoAb and inhibitory peptides are added to serum samples at various concentrations. Diluted samples were added to the plate and incubated overnight at 4 ℃. After 24 hours, the plates were washed thoroughly with wash buffer, then 100. Mu.l 4mM barbital, 145mM NaCl, 2mM CaCl ₂ 、1mM MgCl ₂ 0.1 μg of purified human C4 (obtained as described in Dodds, A.W., methods enzymol.223:46, 1993) at pH 7.4 was added to each well. After 1.5 hours at 37 ℃, the plates were washed again and C4b deposition was detected using alkaline phosphatase conjugated chicken anti-human C4C antibodies (obtained from immunosystem, uppsala, sweden) and measured using colorimetric substrate p-nitrophenylphosphate.

C4 assay on mannan: the above assay is suitable for measuring lectin pathway activation via MBL by coating plates with LSP and mannan prior to addition of serum mixed with various MASP-2 inhibitors.

C4 assay on H-fiber gelling protein (Hakata Ag): the above assay is suitable for measuring lectin pathway activation via H-fiber gelling protein by coating plates with LPS and H-fiber gelling protein prior to addition of serum mixed with various MASP-2 inhibitors.

Example 8

The following assay demonstrates the presence of classical pathway activation in wild-type and MASP-2-/-mice.

The method comprises the following steps: microtiter plates were coated with 0.1% human serum albumin in 10mM Tris, 140mM NaCl,pH 7.4 at room temperatureNunc, catalog number 442404,Fisher Scientific) for a total of 1 hour, followed by a reaction at 4℃with a reaction at TBS/tween/Ca ²⁺ In 1:1000 dilution of sheep anti-whole serum antisera (Scottish Antibody Production Unit, carluke, scotland) were incubated overnight to generate immune complexes in situ. Serum samples were obtained from wild-type and MASP-2-/-mice and added to the coated plates. Control samples were prepared in which C1q was depleted from wild-type and MASP-2/serum samples. According to the specifications of the suppliers,coupling using protein A coated with rabbit anti-human C1q IgG (Dako, glosteup, denmark)>(Dynal Biotech, oslo, norway) to prepare C1q depleted mouse serum. Plates were incubated for 90 minutes at 37 ℃. Bound C3b was detected with polyclonal anti-human C3C antibody (Dako A062) diluted 1:1000 in TBS/tw/Ca++. The secondary antibody is goat anti-rabbit IgG.

Results: FIG. 7 shows the relative C3b deposition levels on IgG coated plates in wild-type serum, MASP-2-/-serum, C1q depleted wild-type and C1q depleted MASP-2-/-serum. These results confirm that the classical pathway is intact in MASP-2-/-mouse strains.

Example 9

By analyzing the effect of MASP-2 inhibitors under conditions in which the classical pathway is initiated by an immune complex, the following assay was used to test whether MASP-2 inhibitors block the classical pathway.

The method comprises the following steps: to test the effect of MASP-2 inhibitors on complement activation conditions in which the classical pathway is initiated by the immune complex, 50 μl samples containing 90% NHS in triplicate were incubated at 37deg.C in the presence of 10 μg/ml Immune Complex (IC) or PBS, and also included in parallel triplicate samples (+/-IC) containing 200nM of the anti-properdin monoclonal antibody during incubation at 37deg.C. After incubation at 37 ℃ for 2 hours, 13mM EDTA was added to all samples to stop further complement activation, and the samples were immediately cooled to 5 ℃. The samples were then stored at 70℃and the complement activation products (C3 a and sC5 b-9) were determined using ELISA kits (Quidel, catalogue No. A015 and A009) following the manufacturer's instructions.

Example 10

This example describes the identification of high affinity anti-MASP-2 Fab2 antibody fragments that block MASP-2 activity.

Background and principle: MASP-2 is a complex protein with many separate functional domains, including: binding site for MBL and fiber gel protein, serine proteinase catalysis site and eggBai Meijie binding site for substrate C2, binding site for proteolytic substrate C4, MASP-2 cleavage site for self-activation of MASP-2 zymogen and two Ca ⁺⁺ Binding sites. Fab2 antibody fragments which bind to MASP-2 with high affinity were identified and the identified Fab2 fragments were tested in a functional assay to determine if they were capable of blocking MASP-2 functional activity.

In order to block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind to and interfere with the structural epitopes on MASP-2 that are essential for MASP-2 functional activity. Thus, many or all of the high affinity binding anti-MASP-2 Fab2 may not inhibit MASP-2 functional activity, except that they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity.

Functional assays measuring inhibition of lectin pathway C3 convertase formation were used to evaluate the "blocking activity" against MASP-2 Fab2. The primary physiological role of MASP-2 in the lectin pathway is known to be the production of the next functional component of the lectin-mediated complement pathway, lectin pathway C3 convertase. Lectin pathway C3 convertases are a key enzymatic complex (C4 bC2 a) that cleaves C3 proteolytically into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4 bC2 a); however, MASP-2 functional activity is required in order to generate the two protein components (C4 b, C2 a) that make up the lectin pathway C3 convertase. Furthermore, all of the separate functional activities of MASP-2 listed above appear to be necessary in order to allow MASP-2 to be used to generate lectin pathway C3 convertases. For these reasons, a preferred assay for assessing "blocking activity" against MASP-2 Fab2 is considered a functional assay that measures inhibition of lectin pathway C3 convertase formation.

Production of high affinity Fab 2: phage display libraries of human variable light and heavy chain antibody sequences, and automated antibody selection techniques for identifying Fab2 reactive with selected ligands of interest, are used to generate high affinity Fab2 against the rat MASP-2 protein (SEQ ID NO: 55). A known amount of rat MASP-2 (> 85% pure) protein was used for antibody screening. Three rounds of amplification were used to select the antibody with the best affinity. Approximately 250 different hits of expressed antibody fragments were selected for ELISA screening. The high affinity hits were then sequenced to determine the uniqueness of the different antibodies.

Fifty unique anti-MASP-2 antibodies were purified and 250 μg of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complement pathway function testing, as described in more detail below.

Assays for assessing inhibition (blocking) activity against MASP-2 Fab2

1. An assay to measure inhibition of lectin pathway C3 convertase formation:

background: lectin pathway C3 convertases are enzymatic complexes (C4 bC2 a) that cleave C3 proteolytically into two powerful pro-inflammatory fragments: anaphylatoxin C3a and opsonin C3b. In terms of mediating inflammation, the formation of C3 convertase appears to be a key step in the lectin pathway. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4 bC2 a); thus, anti-MASP-2 antibodies (or Fab 2) do not directly inhibit the activity of pre-existing C3 convertases. However, MASP-2 serine protease activity is required in order to generate the two protein components (C4 b, C2 a) that make up the lectin pathway C3 convertase. Thus, an anti-MASP-2 Fab2 that inhibits the functional activity of MASP-2 (i.e., a blocking anti-MASP-2 Fab2) will inhibit de novo formation of lectin pathway C3 convertases. C3 contains rare and highly reactive thioester groups as part of its structure. After cleavage of C3 by the C3 convertase in this assay, the thioester group on C3b may form a covalent bond with a hydroxyl or amino group on a macromolecule immobilized via an ester or amide linkage on the bottom of the plastic well, thus facilitating C3b detection in an ELISA assay.

Yeast mannans are known activators of the lectin pathway. In the following method for measuring C3 convertase formation, plastic wells coated with mannan were incubated with diluted rat serum at 37 ℃ for 30 minutes to activate the lectin pathway. The wells were then washed and the C3b immobilized on the wells was determined using standard ELISA methods. The amount of C3b produced in this assay is a direct reflection of de novo formation of lectin pathway C3 convertase. anti-MASP-2 Fab2 at selected concentrations was tested in this assay for its ability to inhibit C3 convertase formation and subsequent C3b production.

The method comprises the following steps:

96-well Costar Medium Binding plates were incubated overnight at 5℃with 1. Mu.g/50. Mu.L/well of mannan diluted in 50mM carbonate buffer, pH 9.5. After overnight incubation, each well was washed 3 times with 200 μl PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated with gentle mixing for one hour at room temperature. Each well was then washed 3 times with 200 μl PBS. anti-MASP-2 Fab2 samples at 5℃in the presence of Ca ⁺⁺ And Mg (magnesium) ⁺⁺ GVB buffer (4.0 mM barbital, 141mM NaCl, 1.0mM MgCl) ₂ 、2.0mM CaCl ₂ 0.1% gelatin, ph 7.4) to a selected concentration. 0.5% rat serum was added to the above samples at 5℃and 100. Mu.L was transferred to each well. Plates were covered and incubated in a 37 ℃ water bath for 30 minutes to allow complement activation. The reaction was terminated by transferring the plates from a 37 ℃ water bath to a vessel containing an ice-water mixture. Each well was washed 5 times with 200 μl PBS Tween 20 (0.05% Tween 20 in PBS) and then twice with 200 μl PBS. mu.L/well of 1:10,000 diluted primary antibody (rabbit anti-human C3C, DAKO A0062) was added to PBS containing 2.0mg/ml bovine serum albumin and incubated with gentle mixing at room temperature for 1 hour. Each well was washed with 5X 200. Mu.L PBS. mu.L/well of 1:10,000 diluted secondary antibody (peroxidase conjugated goat anti-rabbit IgG, american Qualex A102 PU) was added to PBS containing 2.0mg/ml bovine serum albumin and incubated with gentle mixing on a shaker for one hour at room temperature. Each well was washed 5 times with 200. Mu.L PBS. 100. Mu.L/well of peroxidase substrate TMB (Kirkegaard&Perry Laboratories) and incubated at room temperature for 10 minutes. By adding 100. Mu.L/well of 1.0. 1.0M H ₃ PO ₄ To terminate the peroxidase reaction, and to measure OD ₄₅₀ 。

2. Assay to measure inhibition of MASP-2 dependent C4 cleavage

Background: the serine protease activity of MASP-2 is highly specific and only two protein substrates of MASP-2 were identified: c2 and C4. Cleavage of C4 generates C4a and C4b. anti-MASP-2 Fab2 may bind to a structural epitope on MASP-2 that is directly involved in C4 cleavage (e.g., the MASP-2 binding site of C4; the MASP-2 serine protease catalytic site) and thereby inhibit the C4 cleavage functional activity of MASP-2.

Yeast mannans are known activators of the lectin pathway. In the following method for measuring C4 cleavage activity of MASP-2, the mannan coated plastic wells were incubated with diluted rat serum at 37℃for 30 minutes to activate the lectin pathway. Since the primary antibody used in this ELISA assay only recognized human C4, diluted rat serum was also supplemented with human C4 (1.0. Mu.g/ml). The wells were then washed and human C4b immobilized on the wells was determined using standard ELISA methods. The amount of C4b produced in this assay is a measure of MASP-2 dependent C4 cleavage activity. anti-MASP-2 Fab2 at selected concentrations was tested in this assay for its ability to inhibit C4b cleavage.

The method comprises the following steps: 96-well Costar Medium Binding plates were incubated overnight at 5℃with 1.0. Mu.g/50. Mu.L/well of mannan diluted in 50mM carbonate buffer, pH 9.5. Each well was washed 3X with 200. Mu.L PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated with gentle mixing for one hour at room temperature. Each well was washed 3X with 200. Mu.L PBS. anti-MASP-2 Fab2 samples at 5℃in the presence of Ca ⁺⁺ And Mg (magnesium) ⁺⁺ GVB buffer (4.0 mM barbital, 141mM NaCl, 1.0mM MgCl) ₂ 、2.0mM CaCl ₂ 0.1% gelatin, pH 7.4) to the selected concentration. Also included in these samples was 1.0 μg/ml human C4 (Quidel). 0.5% rat serum was added to the above samples at 5℃and 100. Mu.L was transferred to each well. Plates were covered and incubated in a 37 ℃ water bath for 30 minutes to allow complement activation. The reaction was terminated by transferring the plates from a 37 ℃ water bath to a vessel containing an ice-water mixture. Each well was washed with 5X 200. Mu.L PBS-Tween 20 (0.05% Tween 20 in PBS), and each well was then washed 2X with 200. Mu.L PBS. A100. Mu.L/well 1:700 dilution of biotin-conjugated chicken anti-human C4C (Immunsystem AB, uppsala, sweden) was added to PBS containing 2.0mg/ml Bovine Serum Albumin (BSA), And incubated at room temperature for one hour with gentle mixing. Each well was washed with 5X 200. Mu.L PBS. 100. Mu.L/well of 0.1. Mu.g/ml peroxidase conjugated streptavidin (Pierce Chemical # 21126) was added to PBS containing 2.0mg/ml BSA and incubated with gentle mixing on a shaker for one hour at room temperature. Each well was washed with 5X 200. Mu.L PBS. 100. Mu.L/well of peroxidase substrate TMB (Kirkegaard&Perry Laboratories) and incubated at room temperature for 16 minutes. By adding 100. Mu.L/well of 1.0. 1.0M H ₃ PO ₄ To terminate the peroxidase reaction, and to measure OD ₄₅₀ 。

3. Binding assays against rat MASP-2 Fab2 and 'native' rat MASP-2

Background: MASP-2 is typically present in plasma as a MASP-2 dimer complex, which also includes specific lectin molecules (mannose binding protein (MBL) and fiber gellin). Thus, if there is an interest in studying the binding of anti-MASP-2 Fab2 to physiologically relevant forms of MASP-2, it is important to develop binding assays in which the interaction between Fab2 and 'native' MASP-2 in plasma, rather than purified recombinant MASP-2, is used. In this binding assay, the 'native' MASP-2MBL complex from 10% rat serum is first immobilized onto mannan coated wells. The binding affinity of various anti-MASP-2 Fab2 to immobilized 'native' MASP-2 was then investigated using standard ELISA methods.

The method comprises the following steps: 96-well Costar High Binding plates were incubated overnight at 5℃with 1. Mu.g/50. Mu.L/well of mannan diluted in 50mM carbonate buffer, pH 9.5. Each well was washed 3X with 200. Mu.L PBS. Wells were blocked with 100 μl/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated with gentle mixing for one hour at room temperature. 200. Mu.L TBS/Tween/Ca per well ⁺⁺ Washing buffer (Tris buffered saline, 0.05% Tween 20, containing 5.0mM CaCl) ₂ pH 7.4) wash 3X. Preparation on ice in high salt binding buffer (20 mM Tris, 1.0M NaCl, 10mM CaCl) ₂ 0.05% Triton-X100, 0.1% (w/v) bovine serum albumin, pH 7.4)100. Mu.L/well was added and incubated overnight at 5 ℃. 200 mu LTBS/Tween/Ca for wells ⁺⁺ Wash buffer wash 3X. The wells were then washed 2X with 200. Mu.L PBS. A selected concentration of 100. Mu.L/well of anti-MASP-2 Fab2 was added, which was in the presence of Ca ⁺⁺ And Mg (magnesium) ⁺⁺ GVB buffer (4.0 mM barbital, 141mM NaCl, 1.0mM MgCl) ₂ 、2.0mM CaCl ₂ 0.1% gelatin, pH 7.4) and incubated for one hour at room temperature with gentle mixing. Each well was washed with 5X 200. Mu.L PBS. 100. Mu.L/well of HRP conjugated goat anti-Fab 2 (Biogenesis, cat. No. 0500-0099) was added, diluted 1:5000 in PBS solution of 2.0mg/ml bovine serum albumin, and incubated with gentle mixing for one hour at room temperature. Each well was washed with 5X 200. Mu.L PBS. 100. Mu.L/well of peroxidase substrate TMB (Kirkegaard &Perry Laboratories) and incubated at room temperature for 70 minutes. By adding 100. Mu.L/well of 1.0. 1.0M H ₃ PO ₄ To terminate the peroxidase reaction, and to measure OD ₄₅₀ 。

Results:

approximately 250 different Fab2 reactive with high affinity to rat MASP-2 protein were selected for ELISA screening. These high affinity Fab2 were sequenced to determine the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 μg of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complement pathway function testing. The results of this analysis are shown in table 6 below.

Table 6: anti-MASP-2 Fab2 blocking lectin pathway complement activation

As shown in Table 6 above, of the 50 anti-MASP-2 Fab2 tested, 17 Fab2 were identified as MASP-2 blocking Fab2, which strongly inhibited C3 convertase formation, with an IC50 (34% positive hit rate) of 10nM Fab2 or less. 8 of the 17 Fab2 identified had IC50 in the sub nanomolar range. Furthermore, all 17 MASP-2 blocking Fab2 shown in Table 6 gave essentially complete inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. Fig. 8A graphically shows the results of the C3 convertase formation assay for Fab2 antibody #11, which Fab2 antibody #11 is representative of the other Fab2 antibodies tested, the results of which are shown in table 6. This is an important consideration, as it is theoretically possible that even when each MASP-2 molecule is bound by Fab2, a "blocking" of Fab2 may only partially inhibit MASP-2 function.

Although mannans are known activators of the lectin pathway, it is theoretically possible that the presence of anti-mannans antibodies in rat serum may also activate the classical pathway and produce C3b via classical pathway C3 convertases. However, 17 blocking anti-MASP-2 Fab2 listed in this example each inhibited C3b production (> 95%) potently, thus confirming the specificity of the assay for lectin pathway C3 convertases.

Binding assays were also performed on all 17 blocking Fab2 in order to calculate the apparent Kd for each. The binding assays for six blocking Fab2, anti-rat MASP-2 Fab2 and native rat MASP-2 are also shown in Table 6. FIG. 8B graphically illustrates the results of binding assays with Fab2 antibody # 11. Similar binding assays were also performed for other Fab2, the results of which are shown in table 6. In general, the apparent Kd obtained by binding each of the 6 Fab2 species to 'native' MASP-2 corresponds reasonably well to the IC50 for Fab2 in the C3 convertase function assay. There is evidence that MASP-2 undergoes a conformational change from an 'inactive' to an 'active' form following activation of its protease activity (Feinberg et al, EMBO J22:2348-59 (2003); gal et al, J.biol. Chem.280:33435-44 (2005)). In normal rat plasma for the C3 convertase formation assay, MASP-2 exists predominantly in an 'inactive' zymogen conformation. In contrast, in the binding assay, MASP-2 is present as part of a complex with MBL bound to immobilized mannans; thus, MASP-2 is in an 'active' conformation (Petersen et al J.Immunol Methods 257:107-16, 2001). Thus, for each of the 17 blocking Fab2 tested in both functional assays, the exact correspondence between IC50 and Kd is not necessarily expected, as Fab2 binds to a different conformational form of MASP-2 in each assay. However, in addition to Fab2#88, there appears to be reasonably close correspondence between IC50 and apparent Kd for each of the other 16 Fab2 species tested in both assays (see table 6).

Several blocking Fab2 were evaluated for inhibition of MASP-2 mediated C4 cleavage. Figure 8C graphically shows the results of the C4 cleavage assay, showing inhibition with Fab2#41, with ic50=0.81 nM (see table 6). As shown in fig. 9, all Fab2 tested were found to inhibit C4 cleavage with IC50 similar to that obtained in the C3 convertase assay (see table 6).

Although mannans are known activators of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in rat serum could also activate the classical pathway and thereby generate C4b by C1s mediated C4 cleavage. However, several anti-MASP-2 Fab2 have been identified which strongly inhibit C4b production (> 95%), thus confirming the specificity of this assay for MASP-2 mediated C4 cleavage. Like C3, C4 contains rare and highly reactive thioester groups as part of its structure. After cleavage of C4 by MASP-2 in this assay, the thioester group on C4b may form a covalent bond with a hydroxyl or amino group on a macromolecule that is immobilized via an ester or amide bond on the bottom of the plastic well, thus facilitating C4b detection in an ELISA assay.

These studies clearly demonstrate the production of high affinity Fab2 against the rat MASP-2 protein, which Fab2 functionally blocks both C4 and C3 convertase activity, thereby preventing lectin pathway activation.

Example 11

This example describes epitope mapping with respect to several blocking anti-rat MASP-2 Fab2 antibodies, which were generated as described in example 10.

The method comprises the following steps:

as shown in fig. 10, using the pED4 vector, the following proteins, all having an N-terminal 6X His tag, were expressed in CHO cells:

rat MASP-2A, full length MASP-2 protein inactivated by changing serine at the active center to alanine (S613A);

rat MASP-2K, altered to reduce the self-activating (R424K) full length MASP-2 protein;

CUBI-II, an N-terminal fragment of rat MASP-2, which contains only CUBI, EGF-like and CUBII domains; and

CUBI/EGF-like, N-terminal fragment of rat MASP-2, which contains only CUBI and EGF-like domains.

These proteins were purified from the culture supernatant by nickel affinity chromatography as previously described (Chen et al, J.biol. Chem.276:25894-02 (2001)).

Using pTrxFus (Invitrogen), the C-terminal polypeptide (CCPII-SP) containing the CCPII and serine protease domains of rat MASP-2 was expressed in E.coli as a thioredoxin fusion protein. Proteins were purified from cell lysates using a Thiobond affinity resin. Thioredoxin fusion partners were expressed as negative controls from empty pTrxFus.

All recombinant proteins were dialyzed into TBS buffer and their concentration was determined by measuring OD at 280 nm.

Dot blot analysis:

serial dilutions of the 5 recombinant MASP-2 polypeptides described above and shown in fig. 10 (and thioredoxin polypeptides as negative controls for ccpi-serine protease polypeptides) were spotted onto nitrocellulose membranes. The amount of protein spotted ranged from 100ng to 6.4pg, in five times the steps. In the subsequent experiments the amount of spotted protein was reduced from 50ng to 16pg, again in a five-fold step. The membrane was blocked with a 5% nonfat dry milk in TBS (blocking buffer) solution and then with a buffer (containing 5.0mM Ca ²⁺ ) 1.0 μg/ml of anti-MASP-2 Fab2. Using HRP conjugated anti-human Fab (AbD/Serotec; 1/10,000 diluted) and ECL detection kit (Amersham) to detect bound Fab2. A membrane was incubated with polyclonal rabbit anti-human MASP-2Ab (described in Stover et al, JImmunol 163:6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP conjugated goat anti-rabbit IgG (Dako; 1/2,000 dilution).

MASP-2 binding assay

ELISA plates were coated overnight at 4℃with 1.0. Mu.g/well of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0). The wells were blocked with a 1% BSA TBS solution and serial dilutions of anti-MASP-2 Fab2 were added to the wells containing 5.0mM Ca ²⁺ In TBS of (c). Plates were incubated for one hour at RT. In use TBS/tween/Ca ²⁺ After 3 washes, the mixture was added to TBS/Ca ²⁺ HRP conjugated anti-human Fab (AbD/Serotec) diluted 1/10,000 in (r), and plates were incubated for a further hour at RT. The bound antibodies were detected using the TMB peroxidase substrate kit (Biorad).

Results:

the results of the dot blot analysis demonstrating the reactivity of Fab2 with various MASP-2 polypeptides are provided in Table 7 below. The values provided in table 7 indicate the amount of spotted protein required to obtain about half the maximum signal intensity. As shown, all polypeptides (except for the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-human MASP-2 serum produced in rabbits).

Table 7: reactivity with various recombinant rat MASP-2 polypeptides on dot blotting

Fab2 antibody #	MASP-2A	CUBI-II	CUBI/EGF-like	CCPII-SP	Thioredoxin
						40	0.16ng	NR	NR	0.8ng	NR
41	0.16ng	NR	NR	0.8ng	NR
						11	0.16ng	NR	NR	0.8ng	NR
49	0.16ng	NR	NR	>20ng	NR
						52	0.16ng	NR	NR	0.8ng	NR
57	0.032ng	NR	NR	NR	NR
						58	0.4ng	NR	NR	2.0ng	NR
60	0.4ng	0.4ng	NR	NR	NR
						63	0.4ng	NR	NR	2.0ng	NR
66	0.4ng	NR	NR	2.0ng	NR
						67	0.4ng	NR	NR	2.0ng	NR
71	0.4ng	NR	NR	2.0ng	NR
						81	0.4ng	NR	NR	2.0ng	NR
86	0.4ng	NR	NR	10ng	NR
						87	0.4ng	NR	NR	2.0ng	NR
Positive control	<0.032ng	0.16ng	0.16ng	<0.032ng	NR

Nr=no reaction. The positive control antibody was polyclonal anti-human MASP-2 serum produced in rabbits.

All Fab2 reacted with MASP-2A and MASP-2K (data not shown). Most Fab2 recognizes the CCPII-SP polypeptide, but does not recognize the N-terminal fragment. Fab2#60 and fab2#57 are two exceptions. Fab2#60 recognized MASP-2A and CUBI-II fragments, but did not recognize the CUBI/EGF-like polypeptide or the CCPII-SP polypeptide, suggesting that it binds to an epitope in CUBII or spanning the CUBII and EGF-like domains. Fab2#57 recognizes MASP-2A, but does not recognize any of the MASP-2 fragments tested, indicating that such Fab2 recognizes an epitope in CCP 1. Fabs 2#40 and #49 bind only to intact MASP-2A. In the ELISA binding assay shown in FIG. 11, fab2#60 also bound to the CUBI-II polypeptide, albeit with slightly lower apparent affinity.

These findings confirm the identification of unique blocking Fab2 against multiple regions of the MASP-2 protein.

Example 12

This example describes the identification of fully human scFv antibodies using phage display that bind to MASP-2 and inhibit lectin-mediated complement activation while leaving the classical (C1 q-dependent) pathway components of the immune system intact.

Overview:

by screening phage display libraries, fully human high affinity MASP-2 antibodies were identified. The variable light and heavy chain fragments of antibodies are isolated in both scFv and full length IgG formats. Human MASP-2 antibodies may be used to inhibit cellular damage associated with lectin pathway-mediated complement pathway activation while leaving the classical (C1 q-dependent) pathway components of the immune system intact. In some embodiments, the subject MASP-2 inhibitory antibodies have the following characteristics: (a) High affinity for human MASP-2 (e.g., KD of 10nM or less), and (b) inhibition of MASP-2-dependent complement activity in 90% human serum with an IC50 of 30nM or less.

The method comprises the following steps:

full length catalytically inactive expression of MASP-2:

the full length cDNA sequence (SEQ ID NO: 4) of human MASP-2 encoding the human MASP-2 polypeptide with leader sequence (SEQ ID NO: 5) was subcloned into the mammalian expression vector pCI-Neo (Promega) which drives eukaryotic expression under the control of the CMV enhancer/promoter region (described in Kaufman R.J. et al, nucleic Acids Research 19:4485-90, 1991;Kaufman,Methods in Enzymology,185:537-66 (1991).

To produce catalytically inactive human MASP-2A protein, site-directed mutagenesis is performed as described in US2007/0172483, incorporated herein by reference. The PCR products were purified after agarose gel electrophoresis and band preparation and single adenosine overlap was generated using standard tailing procedures. The adenosine-tailed MASP-2A was then cloned into pGEM-T easy vector and transformed into E.coli. Human MASP-2A was further subcloned into the mammalian expression vector pED or pCI-Neo.

The MASP-2A expression construct described above was transfected into DXB1 cells using standard calcium phosphate transfection procedures (Maniatis et al, 1989). MASP-2A was produced in serum-free medium to ensure that the formulation was not contaminated with other serum proteins. Media was harvested from the fused cells every other day (four total). The level of recombinant MASP-2A averaged approximately 1.5mg/L of medium. MASP-2A (Ser-Ala mutant as described above) was purified by affinity chromatography on MBP-A agarose columns

MASP-2A ELISA for ScFv candidate clones identified by panning/scFv conversion and filtration screening

Phage display libraries of human immunoglobulin light and heavy chain variable region sequences were subjected to antigen panning followed by automated antibody screening and selection to identify high affinity scFv antibodies against human MASP-2 proteins. Three rounds of panning of scFv phage libraries against HIS-tagged or biotin-tagged MASP-2A were performed. The third round of panning was first eluted with MBL and then with TEA (alkaline). To monitor the specific enrichment of phages displaying scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was performed. scFv genes from round 3 panning were cloned into the pheg expression vector and run in a small scale filter screen to find specific clones for MASP-2A.

Bacterial colonies containing plasmids encoding scFv fragments from the third round of panning were selected, rasterized (meshed) on nitrocellulose membranes, and grown overnight on non-induction medium to generate master plates. A total of 18,000 colonies were picked and analyzed from the third round of panning, half from competitive elution and half from subsequent TEA elution. Panning of the scFv phagemid library against MASP-2A followed by scFv conversion and filter screening resulted in 137 positive clones. 108/137 clones were positive for MASP-2 binding in ELISA assays (data not shown), 45 of which were further analyzed for the ability to block MASP-2 activity in normal human serum.

Assay to measure inhibition of lectin pathway C3 convertase formation

Functional assays measuring inhibition of lectin pathway C3 convertase formation were used to evaluate the "blocking activity" of anti-MASP-2 scFv candidate clones. MASP-2 serine protease activity is required in order to generate the two protein components (C4 b, C2 a) that make up the lectin pathway C3 convertase. Thus, an anti-MASP-2 scFv that inhibits the functional activity of MASP-2 (i.e., a blocking MASP-2 scFv) will inhibit de novo formation of lectin pathway C3 convertases. C3 contains rare and highly reactive thioester groups as part of its structure. After cleavage of C3 by the C3 convertase in this assay, the thioester group on C3b may form a covalent bond with a hydroxyl or amino group on a macromolecule that is immobilized via an ester or amide bond on the bottom of the plastic well, thus facilitating C3b detection in an ELISA assay.

Yeast mannans are known activators of the lectin pathway. In the following method for measuring C3 convertase formation, plastic wells coated with mannan are incubated with diluted human serum to activate the lectin pathway. The wells were then washed and the C3b immobilized on the wells was determined using standard ELISA methods. The amount of C3b produced in this assay is a direct reflection of de novo formation of lectin pathway C3 convertase. MASP-2scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b production.

The method comprises the following steps:

45 candidate clones identified as described above were expressed, purified and diluted to the same stock concentration, again in GVB buffer (4.0 mM barbital, 141mM NaCl, 1.0mM MgCl) containing Ca++ and Mg++ ₂ 、2.0mM CaCl ₂ 0.1% gelatin, pH 7.4) to ensure that all clones have the same amount of buffer. scFv clones were each tested in triplicate at a concentration of 2 μg/mL. The positive control was OMS100 Fab2 and tested at 0.4. Mu.g/mL. C3C formation was monitored in the presence and absence of scFv/IgG clones.

Mannan was buffered in 50mM carbonate buffer (15 mM Na ₂ CO ₃ +35mM NaHCO ₃ +1.5mM NaN ₃ ) Diluted to a concentration of 20 μg/mL (1 μg/well) in pH 9.5 and coated overnight on ELISA plates at 4 ℃. The next day, the mannan-coated plates were washed 3 times with 200 μl PBS. Mu.l of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room temperature. Plates were washed 3 times with 200 μl PBS and stored on ice with 200 μl PBS until samples were added.

Normal human serum was diluted to 0.5% in camgvb buffer and scFv clones or OMS100 Fab2 positive controls were added in triplicate at 0.01 μg/mL; to this buffer, 1 μg/mL (OMS 100 control only) and 10 μg/mL were added and pre-incubated on ice for 45 min before addition to the blocked ELISA plate. The reaction was started by incubation at 37 ℃ for one hour and terminated by transferring the plates to an ice bath. Goat C3b deposition was detected with rabbit α -mouse C3C antibody followed by goat α -rabbit HRP. The negative control was an antibody-free buffer (no antibody = maximum C3b deposition), and the positive control was a buffer with EDTA (no C3b deposition). The background was determined by performing the same assay except for the mannan-free wells. Background signal for plates without mannan was subtracted from the signal in the wells with mannan. The cut-off criteria were set to half the activity of the unrelated scFv clone (VZV) and buffer alone.

Results:based on the cut-off criteria, a total of 13 clones were found to block MASP-2 activity. Selection and sequencing to generate>All 13 clones pressed by 50% route gave 10 unique clones. All ten clones were found to have the same light chain subclass λ3, but three different heavy chain subclasses: VH2, VH3 and VH6. In the functional assay, using 0.5% human serum, five of the ten candidate scFv clones gave IC50 nM values of less than 25nM target standard.

To identify antibodies with improved potency, three parent scFv clones identified as described above were subjected to light chain shuffling. This process involves the generation of a combinatorial library consisting of VH of each master clone paired with a human lambda light chain (VL) library derived from 6 healthy donors for the first time used in the experiment. scFv clones with improved binding affinity and/or functionality are then screened in the library.

Table 8: functional potency comparison in IC50 (nM) of the leader clone and its respective parent clone (both in scFv form)

The heavy chain variable region (VH) sequences for the parent and child clones shown in table 8 above are presented below.

Kabat CDRs (31-35 (H1), 50-65 (H2), and 95-107 (H3)) are bold; and the Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) are underlined.

17D20_35VH-21N11VL heavy chain variable region (VH) (SEQ ID NO:67, encoded by SEQ ID NO: 66)

d17N9 heavy chain variable region (VH) (SEQ ID NO: 68)

Presented below are the light chain variable region (VL) sequences for the parent and child clones shown in table 8 above.

Kabat CDR (24-34 (L1), 50-56 (L2), and 89-97 (L3) are bold, and Chothia CDR (24-34 (L1)), 50-56 (L2), and 89-97 (L3) are underlined.

17D20m_d3521N11 light chain variable region (VL) (SEQ ID NO:69, SEQ ID NO NO 70 code)

17N16m_d17N9 light chain variable region (VL) (SEQ ID NO: 71)

MASP-2 antibodies OMS100 and MoAb_d3521N11VL (comprising the heavy chain variable region as shown in SEQ ID NO:67 and the light chain variable region as shown in SEQ ID NO:69, also referred to as "OMS646" and "mAb 6") that have been demonstrated to bind to human MASP-2 with high affinity and have the ability to block functional complement activity were analyzed for epitope binding by dot blot analysis. The results show that OMS646 and OMS100 antibodies are highly specific for MASP-2 and do not bind to MASP-1/3. Antibodies bind neither to MAP19 nor to MASP-2 fragments that do not contain the MASP-2 CCP1 domain, resulting in the conclusion that the binding site contains CCP 1.

MASP-2 antibody OMS646 was determined to bind avidly to recombinant MASP-2 (Kd 60-250 pM) with > 5000-fold selectivity when compared to C1s, C1r or MASP-1 (see Table 9 below):

table 9: affinity and specificity of OMS646 MASP-2 antibody-MASP-2 interaction as assessed by solid phase ELISA studies

Antigens	K _D (pM)
		MASP-1	>500,000
MASP-2	62±23*
		MASP-3	>500,000
Purified human C1r	>500,000
		Purified human C1s	～500,000

* Mean ± SD; n=12

OMS646 specifically blocks lectin-dependent activation of terminal complement components

The method comprises the following steps:

the effect of OMS646 on Membrane Attack Complex (MAC) deposition was analyzed using pathway-specific conditions of the lectin pathway, classical pathway and alternative pathway. For this purpose, the Wieslab Comp300 complement screening kit (Wieslab, lund, sweden) was used following the manufacturer's instructions.

Results:

FIG. 12A graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibodies (OMS 646) under lectin pathway specific assay conditions. FIG. 12B graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS 646) under classical pathway-specific assay conditions. FIG. 12C graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS 646) under alternative pathway-specific assay conditions.

As shown in fig. 12A, OMS646 blocks lectin pathway-mediated activation of MAC deposition, IC thereof ₅₀ The value was about 1nM. However, OMS646 had no effect on MAC deposition generated by classical pathway-mediated activation (fig. 12B) or alternative pathway-mediated activation (fig. 12C).

Pharmacokinetics and pharmacodynamics of OMS646 following Intravenous (IV) or Subcutaneous (SC) administration to mice

In a 28-day single dose PK/PD study in mice, the Pharmacokinetics (PK) and Pharmacodynamics (PD) of OMS646 were evaluated. The study tested 5mg/kg and 15mg/kg OMS646 dose levels for Subcutaneous (SC) administration, as well as 5mg/kg OMS646 dose levels for Intravenous (IV) administration.

Regarding PK profile of OMS646, fig. 13 graphically illustrates OMS646 concentration as a function of time (n=3 animals/group mean) after administration of OMS646 at indicated doses. As shown in FIG. 13, OMS646 reached a maximum plasma concentration of 5-6 μg/mL at 5mg/kg SC approximately 1-2 days post-administration. The bioavailability of OMS646 at 5mg/kg SC was approximately 60%. As further shown in FIG. 13, OMS646 reached a maximum plasma concentration of 10-12 μg/mL at 15mg/kg SC, approximately 1 to 2 days post-administration. For all groups, OMS646 cleared slowly from the systemic circulation, with a terminal half-life of about 8-10 days. The profile of OMS646 is typical for human antibodies in mice.

PD activity of OMS646 is shown graphically in fig. 14A and 14B. FIGS. 14A and 14B show PD responses (decrease in systemic lectin pathway activity) for each mouse in the 5mg/kg IV (FIG. 14A) and 5mg/kg SC (FIG. 14B) groups. The dashed line indicates the baseline of the assay (maximum inhibition; first used experimental mouse serum spiked with excess OMS646 in vitro prior to the assay). As shown in fig. 14A, the systemic lectin pathway activity immediately decreased to near undetectable levels following IV administration of 5mg/kg OMS646, and the lectin pathway activity showed only modest recovery over a 28 day observation period. As shown in fig. 14B, a time-dependent inhibition of lectin pathway activity was observed in mice dosed with 5mg/kg OMS646 SC. Lectin pathway activity drops to near undetectable levels within 24 hours of drug administration and remains at low levels for at least 7 days. Lectin pathway activity gradually increased over time, but did not return to pre-dose levels over the 28 day observation period. Lectin pathway activity versus time profile observed after 15mg/kg SC administration, similar to 5mg/kg SC dose (data not shown), indicated saturation of PD endpoint. The data further indicate that a weekly dose of 5mg/kg OMS646 administered in IV and SC is sufficient to achieve continuous suppression of systemic lectin pathway activity in mice.

Example 13

This example describes the generation of recombinant antibodies that inhibit MASP-2, including heavy and/or light chain variable regions comprising one or more cDNAs that specifically bind to MASP-2 and at least one SGMI core peptide sequence (also referred to as MASP-2 antibodies or antigen binding fragments thereof bearing an SGMI peptide).

Background/principle:

the production of MASP-2 specific inhibitors, known as SGMI-2, is described in Heja et al, J Biol Chem 287:20290 (2012) and Heja et al, PNAS 109:10498 (2012), each of which is incorporated herein by reference. SGMI-2 is a 36 amino acid peptide selected from a phage library of variants of desert locust (Schistocerca gregaria) protease inhibitor 2, in which six of the eight positions of the protease binding loop are fully randomized. Subsequent in vitro evolution gave a single nM K _I Monospecific inhibitors of the values (Heja et al J.biol. Chem.287:20290, 2012). Structural research uncoveringIt is shown that the optimized protease binding loop forms the primary binding site, which defines the specificity of both inhibitors. The amino acid sequences of the extended secondary and internal binding regions are common to both inhibitors and contribute to the contact interface (Heja et al 2012.j.biol. Chem. 287:20290). Mechanistically, SGMI-2 blocks the lectin pathway of complement activation without affecting the classical pathway (Heja et al 2012.Proc. Natl. Acad. Sci.109:10498).

The amino acid sequence of the SGMI-2 inhibitor is set forth below:

SGMI-2-full length: LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ(SEQ ID NO:72)

SGMI-2-medium: TCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:73)

SGMI-2-short: … … … … … … … … … … … … TCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:74)

The MASP-2 antibodies and fragments thereof bearing the SGMI-2 peptide are produced by fusing the SGMI-2 peptide amino acid sequence (e.g., SEQ ID NO:72, 73 or 74) to the amino or carboxy terminus of the heavy and/or light chain of a human MASP-2 antibody as described in this example and in WO 2014/144542. As described in WO2014/144542, antibodies and fragments of MASP-2 carrying the SGMI-2 peptide have enhanced inhibitory activity compared to naked MASP-2 scaffold antibodies without the SGMI-2 peptide sequence when measured in a C3b or C4b deposition assay using human serum, and also enhanced inhibitory activity compared to naked MASP-2 scaffold antibodies when measured in an in vivo mouse model. Methods for producing MASP-2 antibodies bearing SGMI-2 peptides are described below.

The method comprises the following steps:

expression constructs were generated to encode four exemplary MASP-2 antibodies bearing an SGMI-2 peptide fused to the N-terminus or C-terminus of the heavy or light chain of a representative MASP-2 inhibitory antibody OMS646 (generated as described in example 12).

Table 10: MASP-2 antibody/SGMI-2 fusion

Abbreviations in table 10:

"H-N" = amino terminus of heavy chain

"H-C" = carboxyl terminus of heavy chain

"L-N" = amino terminus of light chain

"L-C" = carboxyl terminus of light chain

"M2" =masp-2 ab scaffold (representative OMS 646)

For the N-terminal fusion shown in Table 10, a peptide linker ('GTGGGSGSSS' SEQ ID NO: 79) was added between the SGMI-2 peptide and the variable region.

For the C-terminal fusion shown in Table 10, a peptide linker ('AAGGSG' SEQ ID NO: 80) was added between the constant region and the SGMI-2 peptide, and a second peptide "GSGA" (SEQ ID NO: 81) was added at the C-terminus of the fusion polypeptide to protect the C-terminal SGMI-2 peptide from degradation.

The amino acid sequences of the following representative MASP-2 antibodies/SGMI-2 fusions are provided below:

H-M2ab6-SGMI-2-N (SEQ ID NO:75, encoded by SEQ ID NO: 82):

the [491aa protein, aa 1-36 = SGMI-2 (underlined), aa37-46 = linker (italic); aa47-164 = MASP-2ab#6 heavy chain variable region (underlined); aa165-491 = IgG4 constant region with hinge mutations. ]

H-M2ab6-SGMI-2-C (SEQ ID NO:76, encoded by SEQ ID NO: 83):

the heavy chain variable region of [491aa protein, aa1-118 = MASP-2ab#6 (underlined); aa 119-445 = IgG4 constant region with hinge mutation; aa 446-451 = linker 1 (italic); aa 452-487 = SGMI-2; aa488-491 = linker 2 (italic). ]

L-M2ab6-SGMI-2-N (SEQ ID NO:77, encoded by SEQ ID NO: 84):

protein 258aa, aa1-36 = SGMI-2 (underlined); aa37-46 = linker (italic); aa47-152 = MASP-2ab#6 light chain variable region (underlined); aa153-258 = human Ig lambda constant region ]

L-M2ab6-SGMI-2-C (SEQ ID NO:78, encoded by SEQ ID NO: 85):

the light chain variable region of [258aa protein, aa1-106 = MASP-2ab#6 (underlined); aa 107-212 = human igλ constant region; aa 213-218 = 1 st linker; aa219-254 = SGMI-2; aa255-258 = 2 nd linker ]

Functional measurement:

four MASP-2-SGMI-2 fusion antibody constructs were transiently expressed in an Expi293F cell (Invitrogen), purified by protein A affinity chromatography, and tested for inhibition of C3b deposition in 10% normal human serum in a mannan-coated bead assay as described below.

C3b deposition of MASP-2-SGMI-2 fusion was tested in a mannan-coated bead assay

MASP-2-SGMI-2 fusion antibodies were evaluated for lectin pathway inhibition in a C3b deposition assay on mannan-coated beads. Such determination of the degree of activity by flow cytometry provides a ratioHigher resolution is measured. Lectin pathway bead assays were performed as follows: IN carbonate-bicarbonate buffer (pH 9.6), the mannans were adsorbed to polystyrene beads (Bangs Laboratories; fisher, IN, USA) 7. Mu.M IN diameter at 4℃overnight. Beads were washed in PBS and exposed to 10% human serum or 10% serum pre-incubated with antibodies or inhibitors. The serum-bead mixture was incubated for one hour at room temperature with agitation. After serum incubation, the beads were washed and C3b deposition on the beads was measured by detection with anti-C3C rabbit polyclonal antibody (Dako North America; carpinteria, CA, USA), and PE-Cy5 conjugated goat anti-rabbit secondary antibody (Southern Biotech; birmingham, AL, USA). After the staining procedure, the beads were analyzed using a FACSCalibur flow cytometer. The beads were gated as a uniform population using forward and side scatter, and C3b deposition was evident as FL3 positive particles (FL-3 or "FL-3 channel" indicated the 3 rd or red channel on the cytometer). The geometric Mean Fluorescence Intensity (MFI) of the population for each experimental condition was plotted against antibody/inhibitor concentration to assess lectin pathway inhibition.

Calculation of IC using GraphPad PRISM software ₅₀ Values. Specifically, IC is obtained by applying a variable slope (four parameters), a log-nonlinear fit (antibody) to the average fluorescence intensity curve obtained from a cell count assay ₅₀ Values.

The results are shown in Table 11.

Table 11: c3b deposition in 10% human serum (mannan coated bead assay)

Constructs	IC ₅₀ (nM)
		Naked N2 ab (mAb # 6)	≥3.63nM
H-M2-SGMI-2-N	2.11nM
		L-M2-SGMI-2-C	1.99nM
H-M2-SGMI-2-N	2.24nM
		L-M2-SGMI-2-N	3.71nM

Results:

control, MASP-2 "naked" scaffold antibody (mAb # 6) without SGMI, which is inhibitory in this assay, has an IC50 value of > 3.63nM, consistent with the inhibition observed in example 12. Notably, as shown in table 11, all SGMI-2-MASP-2 antibody fusions tested improved the efficacy of MASP-2 scaffold antibodies in this assay, suggesting that increased titers may also be beneficial in the inhibition of C3b deposition.

C4b deposition of MASP-2-SGMI-2 fusion was tested in a mannan-coated bead assay with 10% human serum Product measurement

The C4b deposition assay was performed with 10% human serum using the same assay conditions as described above for the C3b deposition assay, with the modifications described below. Prior to flow cytometry analysis, C4b detection and flow cytometry analysis were performed by staining the deposition reaction with anti-C4 b mouse monoclonal antibody (1:500, quidel) and with a secondary goat anti-mouse F (ab') 2 conjugated to PE Cy5 (1:200,Southern Biotech).

Results:

the SGMI-2-bearing MASP-2-N-terminal antibody fusion (H-M2-SGMI-2-N: ic50=0.34 nM), L-M2-SGMI-2-N: ic50=0.41 nM) has increased potency compared to the MASP-2 scaffold antibody (HL-M2: ic50=0.78 nM).

Similarly, MASP-2 scaffold antibodies (HL-M2: IC ₅₀ =1.2 nM), C-terminal MASP-2 antibody fusion with a single SGMI-2 (H-M2-SGMI-2-C: IC) ₅₀ =0.45 nM and L-M2-SGMI-2c ic ₅₀ =0.47 nM) both have increased potency.

C3b of MASP-2-SGMI-2 fusion was tested in a mannan-coated bead assay with 10% mouse serum And (5) depositing.

Mannan-coated bead assays for C3b deposition were performed with 10% mouse serum as described above. Similar to the results observed in human serum, MASP-2 fusions bearing SGMI-2 were determined to have increased potency compared to MASP-2 scaffold antibodies in mouse serum.

Summarizing the results: the results in this example demonstrate that all SGMI-2-MASP-2 antibody fusions tested improved the efficacy of MASP-2 scaffold antibodies.

Example 14

This example provides results generated using a Unilateral Ureteral Obstruction (UUO) model of renal fibrosis in MASP-2-/-deficient and MASP-2+/+ full mice to assess the role of the lectin pathway in renal fibrosis.

Background/principle:

renal fibrosis and inflammation are the dominant features of advanced kidney disease. Tubular interstitial fibrosis is a progressive process involving sustained cellular injury, abnormal healing, activation of resident and infiltrating kidney cells, cytokine release, inflammation and phenotypic activation of kidney cells to produce extracellular matrix. Tubular Interstitial (TI) fibrosis is a common endpoint of multiple renal pathological conditions and represents a key target for potential therapies aimed at preventing progressive impairment of renal function in Chronic Kidney Disease (CKD). Renal TI injury is closely linked to reduced renal function in glomerular disease (Risdon R.A. et al, lancet 1:363-366, 1968;Schainuck L.I. Et al, hum Pathol 1:631-640, 1970; nath K.A., am J Kid Dis 20:1-17, 1992), and is characteristic of CKD, where there is accumulation of myofibroblasts and potential space between tubules and capillaries surrounding the tubules becomes occupied by matrix composed of collagen and other proteoglycans. The origin of TI myofibroblasts remains strongly controversial, but fibrosis is generally preceded by TI accumulation of T lymphocytes, initially characterized by macrophages (Liu Y. Et al, nat Rev Nephrol 7:684-696, 2011;Duffield J.S., J Clin Invest 124:2299-2306, 2014).

Rodent models of UUO generate progressive renal fibrosis, a marker of progressive renal disease of virtually any etiology (Chevalier et al Kidney International 75:1145-1152, 2009). It has been reported that C3 gene expression is increased in wild-type mice following UUO, and collagen deposition is significantly reduced in C3-/-knockout mice following UO compared to wild-type mice, suggesting a role for complement activation in renal fibrosis (Fearn et al, mol Immunol48:1666-1733, 2011). It has also been reported that in models of tubular interstitial injury, C5 deficiency leads to a significant improvement in the major components of renal fibrosis (Boor P. Et al J of Am Soc of Nephrology:18:1508-1515, 2007). However, prior to the studies described herein by the inventors, the specific complement components involved in renal fibrosis have not been well defined. Thus, the following study was performed to evaluate MASP-2 (-/-) and MASP-2 (+/+) male mice in a Unilateral Ureteral Obstruction (UUO) model.

The method comprises the following steps:

MASP-2-/-mice were generated as described in example 1 and backcrossed to C57BL/6 for 10 passages. Male wild-type (WT) C57BL/6 mice, as well as homozygous MASP-2 deficient (MASP-2-/-) mice in the C57BL/6 background, were kept on standard conditions for 12/12 day/night cycles, fed standard food pellets, and given free access to food and water. Ten week old mice, 6/group, were anesthetized with 2.5% isoflurane in 1.5L/min oxygen. Two groups of ten week old male C56/BL6 mice (wild type and MASP-2-/-) were surgically ligated with right ureters. The right kidney was exposed through a 1cm flank incision. The right ureter was completely obstructed at two points using a 6/0polyglactin suture. Buprenorphine analgesia is provided every 12 hours during the perioperative period, with up to 5 doses depending on pain scores. Local bupivacaine anesthesia is administered once during surgery.

Mice were sacrificed 7 days after surgery and kidney tissue was collected, fixed and embedded in paraffin blocks. Blood was collected from mice under anesthesia by cardiac puncture, and the mice were sacrificed by exsanguination after nephrectomy. Blood was allowed to set on ice for 2 hours and serum was separated by centrifugation and kept frozen as an aliquot at-80 ℃.

Immunohistochemistry of kidney tissue

To measure the extent of renal fibrosis as indicated by collagen deposition, 5 micron paraffin embedded kidney sections were stained with sirius red, a collagen specific stain as described in Whittaker p. Et al Basic Res Cardiol 89:397-410, 1994. Briefly, kidney sections were deparaffinized, rehydrated, and collagen was stained with aqueous sirius red in 500mL of saturated aqueous picric acid (0.5 gm of sirius red, sigma, dorset UK) for 1 hour. Slides were washed twice in acidified water (0.5% glacial acetic acid in distilled water) for 5 minutes each, then dehydrated and fixed.

To measure the degree of inflammation as indicated by macrophage infiltration, kidney sections were stained with macrophage specific antibody F4/80 as follows. Formalin-fixed, paraffin-embedded 5 micron kidney sections were deparaffinized and rehydrated. Antigen retrieval was performed in citrate buffer at 95 ℃ for 20 min, followed by a reaction at 3% H ₂ O ₂ For a period of 10 minutes to quench the endogenous peroxidase activity. Tissue sections were incubated in blocking buffer (10% heat inactivated normal goat serum with 1% bovine serum albumin in Phosphate Buffered Saline (PBS)) for 1 hour at room temperature followed by avidin/biotin blocking. After each step, the tissue sections were washed 3 times in PBS,for a total of 5 minutes. F4/80 macrophage primary antibody (Santa Cruz, dallas, TX, USA) diluted 1:100 in blocking buffer was applied for 1 hour. A 1:200 dilution of biotinylated goat anti-rat secondary antibody was then applied for 30 minutes followed by horseradish peroxidase (HRP) conjugated enzyme for 30 minutes. Staining was performed using Diaminobenzidine (DAB) substrate (Vector Labs, peterborough UK) for 10 minutes, then the slides were washed in water, dehydrated and immobilized without counterstaining to facilitate computer-based analysis.

Image analysis

The percentage of renal cortex staining was determined as described in Furness P.N. et al, J Clin Pathol 50:118-122, 1997. Briefly, 24-bit color images were captured from sequential non-overlapping areas of renal cortex directly beneath the renal capsule around the entire periphery of the renal sections. After each image capture, the NIH image is used to extract the red channel as an 8-bit monochromatic image. The pre-recorded image of the illuminated microscope field without the slice in place is used to subtract the non-uniformities in the background illumination. The image is subjected to a fixed threshold to identify areas of the image corresponding to staining positives. The percentage of black pixels is then calculated and after all images around the kidneys have been measured in this way, the average percentage is recorded, providing a value corresponding to the percentage of stained area in the kidney section.

Gene expression analysis

Expression of several genes associated with kidney inflammation and fibrosis in the kidneys of mice was measured by quantitative PCR (qPCR) as follows. According to the manufacturer's instructions, use(ThermoFisher Scientific, paisley, UK) total RNA was isolated from the renal cortex. The extracted RNA was treated with a Turbo DNA-free kit (ThermoFisher Scientific) to eliminate DNA contamination, and then AMV Reverse Transcription System (Promega, madison, wis., USA) was used to synthesize first strand cDNA. By a single qPCR reaction using TaqMan GAPDH Assay (Applied Biosystems, paisley UK), followed by using CqPCR reactions in ustom TaqMan Array 96-well plates (Life Technologies, paisley, UK) were performed to confirm cDNA integrity.

Twelve genes were studied in this analysis:

collagen IV type alpha 1 (col 4 alpha 1; measuring ID: mm01210125 _m1)

Transforming growth factor beta-1 (TGF beta-1; assay ID: mm01178820 _m1);

cadherin 1 (Cdh 1; assay ID: mm01247357 _m1);

fibronectin 1 (Fn 1; assay ID: mm01256744 _m1);

interleukin 6 (IL 6; assay ID Mm00446191 _m1);

interleukin 10 (IL 10; assay ID Mm00439614 _m1);

interleukin 12a (IL 12a; assay ID Mm00434165 _m1);

Vimentin (Vim; assay ID Mm01333430 _m1);

actin α1 (Actn 1; assay ID Mm01304398 _m1);

tumor necrosis factor-alpha (TNF-alpha; assay ID Mm00443260 _g1)

Complement component 3 (C3; assay ID Mm00437838 _m1);

interferon gamma (Ifn-gamma; determination of ID Mm 01168134)

The following housekeeping control genes were used:

glyceraldehyde-3-phosphate dehydrogenase (GAPDH; assay ID Mm99999915 _g1);

glucuronidase beta (gusbeta; assay ID Mm00446953 _m1);

eukaryotic 18S rRNA (18S; assay ID Hs99999901 _s1);

hypoxanthine guanine phosphoribosyl transferase (HPRT; determination of ID Mm00446968 _m1)

20 μL reactions were amplified for 40 cycles using TaqMan Fast Universal Master Mix (Applied Biosystems). Real-time PCR amplification data was analyzed using Applied Biosystems 7000sds v1.4 software.

Results:

following Unilateral Ureteral Obstruction (UUO), the obstructed Kidney undergoes an influx of inflammatory cells, particularly macrophages, followed by a rapid progression of fibrosis, as evidenced by accumulation of collagen, along with tubular dilation and thinning of the proximal tubular epithelium (see Chevalier r.l. et al, kidney Int75:1145-1152, 2009).

Figure 15 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with sirius red, obtained from wild-type and MASP-2-/-mice 7 days after ureteric obstruction (UUO), or sham operated control mice. As shown in fig. 15, kidney sections of wild-type mice after ureteral obstruction for 7 days showed significantly more collagen deposition (p-value=0.0096) than MASP-2-/-mice. In the wild type and MASP-2-/-groups, the standard errors for the mean ± average of UUO operated mice were 24.79 ± 1.908 (n=6) and 16.58 ± 1.3 (n=6), respectively. As further shown in fig. 15, tissue sections of control wild-type and sham operated control MASP-2-/-mice from sham surgery, as expected, showed very low levels of collagen staining.

FIG. 16 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with F4/80 macrophage specific antibody, wherein the tissue sections were wild-type and MASP-2-/-mice 7 days after ureteric obstruction, or sham operated control mice. As shown in fig. 16, tissue obtained from UUO kidneys from MASP-2-/-mice showed significantly less macrophage infiltration after ureteral obstruction for 7 days compared to wild-type mice (% macrophage area stained in WT: 2.23±0.4 versus MASP-2-/-:0.53±0.06, p=0.0035). As further shown in FIG. 16, tissue sections of wild-type and sham operated MASP-2-/-mice from sham surgery did not show detectable macrophage staining.

Gene expression analysis of various genes associated with nephritis and fibrosis was performed in kidney tissue sections from wild-type and MASP-2-/-mice after 7 days of ureteral obstruction, as well as sham-operated wild-type and MASP-2-/-mice. The data shown in fig. 17-20 are Log10 relative to the relative quantification of wild-type sham surgical samples, and bars represent standard errors of the mean. With respect to the results of gene expression analysis of fibrosis-related genes, FIG. 17 graphically illustrates the relative mRNA expression levels of collagen IV type α1 (collagen-4) in kidney tissue sections obtained from wild-type and MASP-2-/-mice after 7 days of ureteric obstruction, as well as sham operated control mice, as measured by qPCR. FIG. 18 graphically illustrates the relative mRNA expression levels of transforming growth factor beta-1 (TGF beta-1) in kidney tissue sections obtained from wild-type and MASP-2-/-mice after 7 days of ureteric obstruction, as well as sham operated control mice, as measured by qPCR. As shown in fig. 17 and 18, the obstructed kidneys from wild-type mice demonstrated a significant increase in the expression of fibrosis-related genes collagen type IV (fig. 17) and tgfβ -1 (fig. 18) compared to sham-operated kidneys in wild-type mice, confirming that these fibrosis-related genes were induced following UUO injury in wild-type mice, as predicted. In contrast, as further shown in fig. 17 and 18, the obstructed kidneys from MASP-2-/-that underwent UUO damage showed a significant decrease in collagen type IV expression (fig. 17, p=0.0388) and a significant decrease in tgfβ -1 expression (fig. 18, p=0.0174) compared to wild-type mice that underwent UUO damage.

With respect to the results of gene expression analysis of inflammation-related genes, FIG. 19 graphically illustrates the relative mRNA expression levels of interleukin 6 (IL-6) in kidney tissue sections obtained from wild-type and MASP-2-/-mice 7 days after ureteral obstruction, as well as sham operated control mice, as measured by qPCR. FIG. 20 graphically illustrates relative mRNA expression levels of interferon-gamma in kidney tissue sections obtained from wild-type and MASP-2-/-mice after 7 days of ureteric obstruction, as well as sham operated control mice, as measured by qPCR. As shown in fig. 19 and 20, the obstructed kidneys from wild-type mice demonstrated a significant increase in the expression of the inflammation-related genes interleukin-6 (fig. 19) and interferon-gamma (fig. 20) compared to the sham-operated kidneys in wild-type mice, confirming that these inflammation-related genes were induced following UUO injury in wild-type mice. In contrast, as further shown in fig. 19 and 20, the obstructed kidneys from MASP-2-/-subjected to UUO injury showed a significant decrease in interleukin 6 (fig. 19, p=0.0109) and interferon- γ expression (fig. 20, p=0.0182) compared to wild-type mice subjected to UUO injury.

Note that in UUO kidneys obtained from both wild-type and MASP-2-/-mice, gene expression of Vim, actn-1, tnfα, C3, and IL-10 were all found to be significantly up-regulated, with no significant difference in expression levels of these specific genes between wild-type and MASP-2-/-mice (data not shown). In the obstructed kidneys from animals in any group, the gene expression levels of Cdh-1 and IL-12a were unchanged (data not shown).

Discussion:

the UUO model in rodents is recognized as inducing early, active and severe lesions in the obstructed Kidney, with reduced renal blood flow, interstitial inflammation and rapid fibrosis within one to two weeks after the obstruction, and has been widely used to understand the common mechanisms and mediators of inflammation and fibrosis in the Kidney (see, e.g., chevalier r.l., kidney Int 75:1145-1152, 2009; yang H. Et al, drug Discov Today Dis Models 7:13-19, 2010).

The results described in this example demonstrate a significant reduction in collagen deposition and macrophage infiltration in UUO-operated kidneys in MASP-2 (-/-) mice relative to wild-type (+/+) control mice. In MASP-2-/-animals, unexpected results showing a significant reduction in kidney injury, both histologically and at the level of gene expression, demonstrated that the complement-activated lectin pathway significantly contributes to the development of inflammation and fibrosis in the obstructed kidney. While not wishing to be bound by a particular theory, it is believed that the lectin pathway critically contributes to the pathophysiology of fibrotic diseases by triggering and maintaining pro-inflammatory stimuli that chronically exist in the vicious circle in which cell damage drives inflammation, which in turn causes further cell damage, scarring and tissue loss. In view of these results, inhibition or blocking of MASP-2 with inhibitors is expected to have a prophylactic and/or therapeutic effect on inhibition or prevention of renal fibrosis, as well as on inhibition or prevention of fibrosis in general (i.e., independent of tissue or organ).

Example 15

This example describes the efficacy analysis of monoclonal MASP-2 inhibitory antibodies in a Unilateral Ureteral Obstruction (UUO) model (murine model of renal fibrosis).

Background/principle:

the improvement of tubular interstitial fibrosis, a common endpoint of multiple renal pathology, represents a key target for therapeutic strategies aimed at preventing progressive renal disease. In view of the lack of new and existing therapies targeting the inflammatory pro-fibrotic pathway in renal disease, there is an urgent need to develop new therapies. Many patients with proteinuria kidney disease show tubular interstitial inflammation and progressive fibrosis, which is closely parallel to reduced kidney function. Proteinuria itself induces the development of tubular interstitial inflammation and proteinuria nephropathy (Brunskill N.J. et al, J Am Soc Nephrol 15:504-505, 2004). Regardless of the primary kidney disease, tubular interstitial inflammation and fibrosis is often seen in patients with progressive kidney impairment and is closely associated with reduced excretory function (Risdon r.a. et al, lancet 1:363-366, 1968;Schainuck L.I. Et al, hum Pathol 1:631-640, 1970). Therapies with the potential to interrupt key common cellular pathways leading to fibrosis are promising for wide-ranging application in renal disease.

As described in example 14, in the UUO model of non-proteinogenic renal fibrosis, MASP-2-/-mice were determined to exhibit significantly less renal fibrosis and inflammation as shown by inflammatory cell infiltration (75% reduction) and histological markers of fibrosis such as collagen (one-third reduction) compared to wild-type control animals, establishing a key role for the lectin pathway in renal fibrosis.

Monoclonal MASP-2 antibodies (OMS 646-SGMI-2 fusions comprising SGMI-2 peptides fused to the C-terminus of OMS646 heavy chain) were generated that specifically block human lectin pathway function, and have also been shown to block mouse lectin pathway, as described in example 13. In this example, OMS646-SGMI-2 was analyzed in a UFO mouse model of kidney fibrosis in wild type mice to determine whether a specific inhibitor of MASP-2 was able to inhibit kidney fibrosis.

The method comprises the following steps:

this study assessed the effect of MASP-2 inhibitory antibodies (10 mg/kg OMS 646-SGMI-2) in male WT C57BL/6 mice compared to human IgG4 isotype control antibody (10 mg/kg ET 904) and vehicle control. Antibodies (10 mg/kg) were administered to groups of 9 mice by intraperitoneal (ip) injection on days 7, 4 and 1 prior to UUO surgery, and again on day 2 post-surgery. Blood samples were taken prior to antibody administration and at the end of the experiment to assess the functional activity of the lectin pathway.

UUO surgery, tissue collection, staining with sirius red and macrophage specific antibody F4/80 was performed using the procedure described in example 14.

The hydroxyproline content of the kidneys of the mice was measured using a specific colorimetric assay test kit (Sigma) according to the manufacturer's instructions.

To evaluate the pharmacodynamic effects of MASP-2 inhibitory mAbs in mice, the systemic lectin pathway activity was assessed by quantifying lectin-induced C3 activation in minimally diluted serum samples collected at indicated times after i.p. administration of MASP-2 mAb or control mAb to mice. Briefly, 7. Mu.M diameter polystyrene microspheres (Bangs Laboratories, fisher IN, USA) were coated with mannan by overnight incubation with 30. Mu.g/mL mannan (Sigma) IN sodium bicarbonate buffer (pH 9.6), then washed, blocked with 1% fetal bovine serum IN PBS, and incubated at 1X10 ⁸ The final concentration of individual beads/mL was resuspended in PBS. Complement deposition reactions were initiated by adding 2.5 μl of mannan-coated beads (-250,000 beads) to 50 μl of minimally diluted mouse serum samples (90% final serum concentration), followed by 40 min incubation at 4 ℃. After the sedimentation reaction was stopped by adding 250. Mu.L of ice-cold flow cytometry buffer (FB: PBS containing 0.1% fetal bovine serum), the beads were collected by centrifugation and washed twice more with 300. Mu.L of ice-cold FB.

To quantify lectin-induced C3 activation, beads were incubated with 50 μl of rabbit anti-human C3C antibody (Dako, carpenter ia, CA, USA) diluted in FB for 1 hour at 4 ℃. After washing twice with FB to remove unbound material, the beads were incubated with 50 μl of goat anti-rabbit antibody conjugated to PE-Cy5 (Southern Biotech, birmingham, AL, USA) diluted in FB for 30 min at 4 ℃. After washing twice with FB to remove unbound material, the beads were resuspended in FB and analyzed by FACS Calibur cytometry. Beads were gated as a uniform population using forward and side scatter, and C3b deposition in each sample was quantified as Mean Fluorescence Intensity (MFI).

Results:

evaluation of collagen deposition:

figure 21 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with sirius red obtained from wild-type mice treated with MASP-2 inhibitory antibodies or isotype control antibodies 7 days after ureteric obstruction. As shown in fig. 21, tissue sections from kidneys harvested 7 days after obstruction (UUO), which were obtained from wild-type mice treated with MASP-2 inhibitory antibodies, showed a significant reduction in collagen deposition (p=0.0477) compared to the amount of collagen deposition in tissue sections from obstructed kidneys, which were obtained from wild-type mice treated with IgG4 isotype control.

Evaluation of hydroxyproline content:

hydroxyproline was measured in kidney tissue as an indicator of collagen content. Hydroxyproline is a parameter that is highly indicative of pathophysiological progression of the disease induced in this model.

Figure 22 graphically illustrates hydroxyproline content from kidneys harvested 7 days after obstruction (UUO) from wild-type mice treated with MASP-2 inhibitory antibodies or IgG4 isotype control. As shown in figure 22, the obstructed kidney tissue from mice treated with MASP-2 inhibitory antibodies demonstrated significantly less hydroxyproline-an indicator of collagen content (p= 0.0439) than the kidney from mice treated with IgG4 isotype control mAb.

Inflammation evaluation:

clear infiltration of macrophages was confirmed by obstructed kidneys from wild-type, isotype control antibody-treated animals and wild-type animals treated with MASP-2 inhibitory antibodies. Careful quantification did not reveal a significant difference in the percentage of macrophage staining area between the two groups (data not shown). However, despite the equivalent number of infiltrating macrophages, the obstructed kidney from MASP-2 inhibitory antibody injected animals showed significantly less fibrosis than the obstructed kidney from isotype control injected animals as judged by sirius red staining, consistent with the following: obstructed kidney tissue from mice treated with MASP-2 inhibitory antibodies had significantly less hydroxyproline than kidneys treated with an IgG4 isotype control mAb.

Discussion of the invention

The results described in this example demonstrate that the use of MASP-2 inhibitory antibodies provides protection against renal fibrosis in the UUO model, which is consistent with the results described in example 14, demonstrating that MASP-2-/-mice have significantly reduced renal fibrosis and inflammation in the UUO model compared to wild-type mice. The results in this example show reduced fibrosis in mice treated with MASP-2 inhibitory antibodies. The discovery of reduced fibrosis is a very significant new discovery in UUO kidneys in animals with reduced or blocked MASP-2 dependent lectin pathway activity. Taken together, the results presented in example 14 and this example demonstrate the beneficial effects of MASP-2 inhibition on tubular interstitial inflammation, tubular cell injury, pro-fibrotic cytokine release and scarring. Alleviation of renal fibrosis remains a key goal of renal therapeutics. The UUO model is a serious model for accelerating renal fibrosis, and interventions in this model that reduce fibrosis, such as the use of MASP-2 inhibitory antibodies, are likely to be used to inhibit or prevent renal fibrosis. The results from the UUO model are likely to be transferable to renal diseases characterized by glomerular and/or proteinuria tubular injury.

Example 16

This example provides results generated using the protein overload proteinuria model of renal fibrosis, inflammation and tubular interstitial injury in MASP-2-/-and wild-type mice to assess the role of the lectin pathway in proteinuria nephropathy.

Background/principle:

proteinuria is a risk factor for the development of renal fibrosis and loss of renal excretion function regardless of the primary renal disease (Tryggvason K. Et al, J International Med 254:216-224, 2003, williams M., am J. Nephrol 25:77-94, 2005). The concept of proteinuria nephropathy describes the toxic effects of excess protein entering the proximal tubules due to impaired glomerular permeation selectivity (Brunskill n.j., J Am Soc Nephrol 15:504-505, 2004, baines r.j., nature Rev Nephrol 7:177-180, 2011). This phenomenon, common to many glomerular diseases, results in a pro-inflammatory scarring environment in the kidney and is characterized by changes in proximal tubule cell growth, apoptosis, gene transcription, and inflammatory cytokine production due to deregulation of the signaling pathway stimulated by the proteinuria tubule fluid. Proteinuria nephropathy is generally recognized as a key contributor to progressive kidney injury common to many primary renal diseases.

Chronic kidney disease affects greater than 15% of the adult population in america and is responsible for approximately 750,000 deaths worldwide per year (Lozano r et al, lancet volume 380, issue 9859:2095-2128, 2012). Proteinuria is an indicator of chronic kidney disease and a factor that contributes to disease progression. Many patients with proteinuria kidney disease show tubular interstitial inflammation and progressive fibrosis, which is closely parallel to reduced kidney function. Proteinuria itself induces the development of tubular interstitial inflammation and proteinuria nephropathy (Brunskill N.J. et al, J Am Soc Nephrol 15:504-505, 2004). In proteinuria kidney disease, excess albumin and other macromolecules are filtered through the glomeruli and are resorbed by the proximal tubular epithelial cells. This causes inflammatory malignancy cycles mediated by complement activation, resulting in cytokine and leukocyte infiltration, which causes tubular interstitial damage and fibrosis, exacerbating proteinuria and resulting loss of kidney function, and eventually progression to end-stage renal failure (see, e.g., clark et al Canadian Medical Association Journal 178:173-175, 2008). Therapies that regulate this deleterious circulation of inflammation and proteinuria are expected to improve outcome in chronic kidney disease.

In view of the beneficial effects of MASP-2 inhibition in the UUO model of tubular interstitial injury, the following experiments were performed to determine whether MASP-2 inhibition reduces kidney injury in the protein overload model. As described in Ishola et al, european Renal Association, 21:591-597, 2006, this study employed protein overload to induce proteinuria kidney disease.

The method comprises the following steps:

MASP-2-/-mice were generated as described in example 1 and backcrossed to BALB/c for 10 passages. Current studies compare the results of wild-type and MASP-2-/-BALB/c mice in the protein overload proteinuria model as follows.

One week prior to the experiment, mice were subjected to unilateral nephrectomy prior to protein overload challenge in order to observe optimal response. The proteinuria inducing agent used was low endotoxin bovine serum albumin (BSA, sigma) given i.p. to WT (n=7) and MASP-2-/-mice (n=7) in normal saline at the following doses: one dose of each of 2mg BSA/gm, 4mg BSA/gm, 6mg BSA/gm, 8mg BSA/gm, 10mg BSA/gm and 12mg BSA/gm body weight, and 9 doses of 15mg BSA/gm body weight, for a total of 15 i.p. administered doses over a 15 day period. Control WT (n=4) and MASP-2-/- (n=4) mice received i.p-administered saline alone. After the last dose was administered, animals were switched in metabolic cages for 24 hours to collect urine. Blood was collected by cardiac puncture under anesthesia, allowed to coagulate on ice for 2 hours, and serum was separated by centrifugation. Serum and urine samples were collected at the end of the experiment on day 15, stored and frozen for analysis.

Mice were sacrificed 24 hours after the last BSA administration at day 15 and various tissues were collected for analysis. Kidneys were harvested and processed for H&E and immunostaining. Immunohistochemical staining was performed as follows. Formalin-fixed, paraffin-embedded 5-micron kidney tissue sections from each mouse were deparaffinized and rehydrated. Antigen retrieval was performed in citrate buffer at 95 °c20 minutes, followed by 3% H in the tissue ₂ O ₂ Incubate for 10 minutes. The tissues were then incubated in blocking buffer with 10% avidin solution (10% serum from the species in which the secondary antibody was produced and 1% bsa in PBS) for 1 hour at room temperature. After each step, the sections were washed 3 times in PBS for 5 minutes each. The primary antibody was then applied in blocking buffer with 10% biotin solution for 1 hour at a concentration of 1:100 for antibodies F4/80 (Santa Cruz catalog #sc-25830), TGF beta (Santa Cruz catalog #sc-7892), IL-6 (Santa Cruz catalog #sc-1265) and 1:50 for TNFa antibodies (Santa Cruz catalog #sc-1348). The biotinylated secondary antibody was then applied for 30 minutes at a concentration of 1:200 for F4/80, TGF beta and IL-6 sections and 1:100 for TNFalpha sections followed by an additional 30 minutes for HRP conjugated enzyme. The color was developed using Diaminobenzidine (DAB) substrate kit (Vector labs) for 10 minutes and the slides were washed in water, dehydrated and immobilized without counterstaining to facilitate computer-based analysis. Stained tissue sections from renal cortex were analyzed by digital image capture followed by quantification using automated image analysis software.

Apoptosis in tissue sections was assessed by staining with terminal deoxynucleotidyl transferase dUTP notch end marker (TUNEL) as follows. Used as followsThe Peroxidase kit (Millipore) stains apoptotic cells in kidney sections. Paraffin-embedded, formalin-fixed kidney sections from each mouse were deparaffinized, rehydrated, and then protein permeabilized with proteinase K (20 μg/mL) applied for 15 minutes to each sample at room temperature. Samples were washed in PBS between steps. By bringing the tissue to 3% H ₂ O ₂ Incubate for 10 minutes to quench endogenous peroxidase activity. The tissue was then incubated in equilibration buffer followed by 1 hour incubation with TdT enzyme at 37 ℃. After washing in stop/wash buffer for 10 min, the anti-digoxigenin conjugate was applied at room temperature for 30 min, followingThe latter is washing. Color development was performed in DAB substrate kit for 4 minutes followed by washing in water. Tissues were counterstained in hematoxylin and fixed in DBX. TUNEL stained (brown stained) apoptotic cells were counted manually in 20 high power fields from a continuous selection of cortex using Leica DBXM optical microscopy.

Results:

evaluation of proteinuria

To confirm the presence of proteinuria in mice, total protein in the serum was analyzed on day 15, and total excreted protein in the urine was measured on day 15 of the study in urine samples collected over a 24 hour period.

Figure 23 graphically shows total serum protein (mg/ml) measured on day 15 in wild-type control mice (n=2) receiving only saline, wild-type mice (n=6) receiving BSA, and MASP-2-/-mice (n=6) receiving BSA. As shown in figure 23, administration of BSA increased serum total protein levels in both wild-type and MASP-2-/-groups to twice the concentration of the saline-only control group, with no significant differences between the treated groups.

Figure 24 graphically illustrates total amount of excreted protein (mg) in urine collected over a 24 hour period on day 15 from wild type control mice (n=2) receiving only saline, wild type (n=6) receiving BSA, and MASP-2-/-mice (n=6) receiving BSA. As shown in fig. 24, at day 15 of this study, there was an approximately six-fold increase in total excreted protein in urine in the BSA-treated group compared to the sham-treated control group that received only saline. The results shown in figures 23 and 24 confirm the working of the proteinuria model as expected.

Evaluation of histological changes in kidneys

Fig. 25 shows representative H & E stained kidney tissue sections harvested from the following groups of mice at day 15 of the protein overload study: (Panel A) wild-type control mice; (Panel B) MASP-2-/-control mice, (Panel C) wild-type mice treated with BSA; and (Panel D) MASP-2-/-mice treated with BSA. As shown in FIG. 25, under the same level of protein overload challenge, there was a much higher degree of tissue preservation in the MASP-2-/-overload group (panel D) than in the wild-type overload group (panel C). For example, the bowden capsule of wild-type mice treated with BSA (overloaded) was observed to be greatly expanded (panel C) compared to the bowden capsule in wild-type control group (panel a). In contrast, bob's vesicles (panel D) in MASP-2-/-mice treated with the same level of BSA (overloaded) retained morphology similar to MASP-2-/-control mice (panel B) and wild-type control mice (panel A). As further shown in FIG. 25, in the proximal and distal tubules of the wild-type kidney section (Panel C), larger protein tubular structures have accumulated, which are larger and richer than MASP-2-/-mice (Panel D).

It should also be noted that analysis of kidney sections from this study by transmission electron microscopy showed that mice treated with BSA had global damage to the ciliary boundaries of distal and proximal tubule cells with cell contents and nuclei protruding into the lumen of the tubule. In contrast, tissues were preserved in MASP-2-/-mice treated with BSA.

Macrophage infiltration evaluation in kidneys

To measure the extent of inflammation as indicated by macrophage infiltration, tissue sections of harvested kidneys were also stained with macrophage specific antibody F4/80 using the method described in Boor et al, J of Am Soc of Nephrology 18:1508-1515, 2007.

Figure 26 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with macrophage specific antibody F4/80, showing the mean staining area (%) of macrophages, wherein the tissue sections were obtained from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2-/-mice treated with BSA (n=5) on day 15 of the protein overload study. As shown in fig. 26, kidney tissue sections stained with F4/80 anti-macrophage antibody showed that while both groups treated with BSA showed a significant increase in kidney macrophage infiltration (measured as% F4/80 antibody staining area) compared to wild-type sham control, a significant decrease in macrophage infiltration was observed in tissue sections from BSA treated MASP-2-/-mice compared to macrophage infiltration in tissue sections from BSA treated wild-type mice (p-value = 0.0345).

Figure 27A graphically illustrates an analysis of the presence of macrophage-proteinuria correlation in each wild-type mouse treated with BSA (n=6) by plotting total excreted protein measured in urine from 24 hours samples against macrophage infiltration (average stained area%). As shown in fig. 27A, most samples from wild-type kidneys showed a positive correlation between the level of proteinuria present and the degree of macrophage infiltration.

Figure 27B graphically illustrates an analysis of the presence of macrophage-proteinuria correlation in each MASP-2-/-mouse treated with BSA (n=5) by plotting total excreted protein in urine of 24 hour samples versus macrophage infiltration (average stained area%). As shown in FIG. 27B, no positive correlation between proteinuria levels and macrophage infiltration levels was observed in wild-type mice in MASP-2-/-mice (shown in FIG. 27A). While not wishing to be bound by any particular theory, these results may indicate the presence of an inflammatory clearance mechanism at high proteinuria levels in MASP-2-/-mice.

Evaluation of cytokine infiltration

Interleukin 6 (IL-6), transforming growth factor beta (TGF beta) and tumor necrosis factor alpha (TNF alpha) are pro-inflammatory cytokines that are known to be up-regulated in wild-type mouse proximal tubules in a model of proteinuria (Abbate M. Et al Journal of the American Society of Nephrology: JASN,17:2974-2984, 2006; david S. Et al, nephrology, didalysis, transplating, official Publication of the European Dialysis and Transplant Association-European Renal Association: 51-56, 1997). Tissue sections of the kidneys were stained with cytokine-specific antibodies as described above.

Figure 28 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tgfβ antibodies (measured as% tgfβ antibody staining area) in wild-type mice treated with BSA (n=4) and MASP-2-/-mice treated with BSA (n=5). As shown in fig. 28, a significant increase in tgfβ staining was observed in the wild-type BSA treated (overloaded) group compared to the MASP-2-/-BSA treated (overloaded) group (p=0.026).

Figure 29 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tnfα antibodies (measured as% tnfα antibody staining area) in wild type mice treated with BSA (n=4) and MASP-2-/-mice treated with BSA (n=5). As shown in fig. 29, a significant increase in tnfα staining was observed in the wild-type BSA treated (overloaded) group compared to the MASP-2-/-BSA treated (overloaded) group (p=0.0303).

Figure 30 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as% IL-6 antibody staining area) in wild type control mice, MASP-2-/-control mice, wild type mice treated with BSA (n=7), and MASP-2-/-mice treated with BSA (n=7). As shown in fig. 30, a highly significant increase in IL-6 staining was observed in the wild-type BSA treated group compared to the MASP-2-/-BSA treated group (p=0.0016).

Apoptosis evaluation

Apoptosis in tissue sections was assessed by staining with terminal deoxynucleotidyl transferase dUTP notch end marker (TUNEL), and the frequency of TUNEL stained apoptotic cells was counted in 20 High Power Fields (HPFs) selected consecutively from the cortex.

Figure 31 graphically illustrates the frequency of TUNEL apoptotic cells counted in 20 High Power Fields (HPF) selected consecutively from tissue sections from renal cortex in wild type control mice (n=1), MASP-2-/-control mice (n), BSA treated wild type mice (n=7) and BSA treated MASP-2-/-mice (n=7). As shown in fig. 31, a significantly higher rate of apoptosis in the cortex was observed in kidneys obtained from wild-type mice treated with BSA (p=0.0001) compared to kidneys obtained from MASP-2-/-mice treated with BSA.

Overall summary of results and conclusions:

the results in this example demonstrate that MASP-2-/-mice have reduced kidney injury in the protein overload model. Thus, inhibitors of MASP-2, such as MASP-2 inhibitory antibodies, are expected to inhibit or prevent inflammation and the deleterious circulation of proteinuria, and to improve outcome in chronic kidney disease.

Example 17

This example describes the analysis of monoclonal MASP-2 inhibitory antibodies with respect to efficacy in reducing and/or preventing renal inflammation and tubular interstitial damage in a mouse protein overload proteinuria model in wild-type mice.

Background/principle:

as described in example 16, in the protein overload model of proteinuria, MASP-2-/-mice were determined to exhibit significantly better results (e.g., less tubular interstitial damage and less kidney inflammation) than wild-type mice, suggesting a pathogenic role for the lectin pathway in proteinuria kidney disease.

As described in example 13, monoclonal MASP-2 inhibitory antibodies (OMS 646-SGMI-2) were generated that specifically block the function of the human lectin pathway and also were shown to block the lectin pathway in mice. In this example, the efficacy of the MASP-2 inhibitory antibody OMS646-SGMI-2 in reducing and/or preventing kidney inflammation and tubular interstitial injury in wild type mice was analyzed in a mouse protein overload proteinuria model.

The method comprises the following steps:

this study assessed the effect of MASP-2 inhibitory antibodies (10 mg/kg OMS 646-SGMI-2) compared to human IgG4 isotype control antibody ET904 (10 mg/kg) and saline control.

Similar to the study described in example 16, this study utilized protein overload to induce proteinuria kidney disease (Ishola et al European Renal Association 21:591-597, 2006). Proteinuria was induced in unilaterally nephrectomized Balb/c mice by daily i.p. injection with increasing doses (2 g/kg to 15 g/kg) of low endotoxin Bovine Serum Albumin (BSA) for a total of 15 days, as described in example 16.

Antibody treatment was administered by i.p. injection every two weeks, starting 7 days prior to proteinuria induction, and continued throughout the study. This dosing regimen was chosen based on previous PK/PD and pharmacological studies, confirming sustained lectin pathway suppression (data not shown). Mice were sacrificed on day 15 and kidneys were harvested and processed for H & E and immunostaining. Stained tissue sections from renal cortex were analyzed by digital image capture followed by quantification using automated image analysis software.

Immunohistochemical staining and apoptosis evaluation were performed as described in example 16.

Results:

evaluation of proteinuria

To confirm the presence of proteinuria in mice, total excreted protein in the urine was measured on day 15 (end of the experiment) in urine samples collected over a 24 hour period. It was determined that urine samples showed an average of almost six-fold increase in total protein levels in the BSA treated group compared to the untreated control group (data not shown), confirming the presence of proteinuria in BSA treated mice. No significant differences were observed in protein levels between BSA treated groups.

Evaluation of histological Change

Fig. 32 shows representative H & E stained tissue sections from the following mice groups on day 15 after treatment with BSA: (Panel A) wild-type control mice treated with saline, (Panel B) control mice treated with isotype antibodies, and (Panel C) wild-type mice treated with MASP-2 inhibitory antibodies.

As shown in figure 32, at the same level of protein overload challenge, there was a much higher degree of tissue preservation in the MASP-2 inhibitory antibody treated group (panel C) compared to the wild type group treated with either saline (panel a) or isotype control (panel B).

Apoptosis evaluation

Apoptosis in tissue sections was assessed by staining with terminal deoxynucleotidyl transferase dUTP notch end marker (TUNEL), and the frequency of TUNEL stained apoptotic cells was counted in 20 High Power Fields (HPFs) selected consecutively from the cortex. Figure 33 graphically illustrates the frequency of TUNEL apoptotic cells counted in 20 High Power Fields (HPF) selected consecutively from tissue sections from renal cortex in wild-type mice treated with saline control and BSA (n=8), wild-type mice treated with isotype control antibodies and BSA (n=8), and wild-type mice treated with MASP-2 inhibitory antibodies and BSA (n=7). As shown in fig. 33, a highly significant reduction in the rate of apoptosis of the cortex was observed in kidneys obtained from the MASP-2 inhibitory antibody treated group compared to the saline and isotype control treated group (p=0.0002 for the saline control and p=0.0052 for the isotype control relative to the MASP-2 inhibitory antibody).

Evaluation of cytokine infiltration

In kidney tissue sections obtained in this study, interleukin 6 (IL-6), transforming growth factor beta (tgfβ) and tumor necrosis factor alpha (tnfα), which are pro-inflammatory cytokines known to be up-regulated in wild-type mouse proximal tubules in the proteinuria model, were evaluated.

Figure 34 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tgfβ antibodies (measured as% tgfβ antibody staining area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibodies (n=7), and BSA and MASP-2 inhibitory antibodies (n=8). As shown in fig. 34, quantification of tgfβ staining regions showed a significant decrease in tgfβ levels (p-value = 0.0324 and 0.0349, respectively) in MASP-2 inhibitory antibody treated mice compared to saline and isotype control antibody treated control groups.

Figure 35 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-tnfα antibodies (measured as% tnfα antibody staining area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibodies (n=7), and BSA and MASP-2 inhibitory antibodies (n=8). As shown in fig. 35, analysis of stained sections showed a significant decrease in tnfα levels in the MASP-2 inhibitory antibody treated group compared to the saline control group (p=0.011) and the isotype control group (p=0.0285).

Figure 36 graphically illustrates the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as% IL-6 antibody staining area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibodies (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibodies (n=8). As shown in fig. 36, analysis of stained sections showed a significant decrease in IL-6 levels in the MASP-2 inhibitory antibody treated group compared to the saline control group (p=0.0269) and the isotype control group (p=0.0445).

Overall summary of results and conclusions:

the results in this example demonstrate that the use of MASP-2 inhibitory antibodies provides protection against kidney injury in a protein overload model, which is consistent with the results described in example 16, demonstrating that MASP-2-/-mice have reduced kidney injury in a proteinuria model.

Example 18

This example provides the results of the use of a kidney disease model generation of doxorubicin-induced kidney fibrosis, inflammation and tubular interstitial injury in MASP-2-/-and wild-type mice to assess the role of the lectin pathway in doxorubicin-induced kidney disease.

Background/principle:

doxorubicin is an anthracycline antitumor antibiotic used in the treatment of a wide variety of cancers, including hematological malignancies, soft tissue sarcomas, and many types of cancers. Doxorubicin-induced nephropathy is a well established rodent model of chronic kidney disease that has enabled a better understanding of the progression of chronic proteinuria (Lee and Harris, nephrology,16:30-38, 2011). The type of structural and functional impairment in doxorubicin-induced kidney disease is very similar to chronic proteinuria kidney disease in humans (Pippin et al American Journal of Renal Physiology 296: F213-29, 2009).

Doxorubicin-induced kidney disease is characterized by injury to podocytes followed by glomerulosclerosis, tubular interstitial inflammation and fibrosis. In many studies doxorubicin-induced nephropathy has been shown to be regulated by both immune and non-immune derived mechanisms (Lee and Harris, nephrology,16:30-38, 2011). Doxorubicin-induced nephropathy has several advantages as a model of kidney disease. First, it is a highly reproducible and predictable model of kidney injury. This is because it is characterized by kidney injury induction within days of drug administration, which allows for simplified experimental design, as the timing of injury is consistent. It is also a model in which the extent of tissue damage is severe, while correlating with acceptable mortality (< 5%) and morbidity (weight loss). Thus, due to the severity and timing of kidney injury in doxorubicin-induced kidney disease, it is a model suitable for testing interventions that protect against kidney injury.

As described in examples 16 and 17, MASP-2-/-mice and mice treated with MASP-2 inhibitory antibodies were determined to show significantly better results (e.g., less tubulointerstitial damage and less nephritis) than wild-type mice in a protein overload model of proteinuria, suggesting a pathogenic role for the lectin pathway in proteinuria kidney disease.

In this example, MASP-2-/-mice were analyzed in a doxorubicin-induced kidney disease model (AN) compared to wild-type mice to determine whether MASP-2 deficiency reduced and/or prevented doxorubicin-induced kidney inflammation and tubular interstitial injury.

The method comprises the following steps:

1. dose and time point optimization

Initial experiments were performed to determine the dose of doxorubicin and the time point at which BALB/c mice developed nephritis levels suitable for testing therapeutic intervention.

Three groups of wild-type BALB/c mice (n=8) were injected with IV-administered single dose of doxorubicin (10.5 mg/kg). Mice were sacrificed at three time points: one week, two weeks and four weeks after doxorubicin administration. Control mice were injected with saline alone.

Results: such as by H&All mice in the three groups showed signs of glomerulosclerosis and proteinuria, as determined by E staining, with increasing levels of tissue inflammation as measured by macrophage infiltration in the kidneys (data not shown). The extent of tissue damage was mild in the one week group, moderate in the two week group, and severe in the four week group (data not shown). Two week time points were selected for the remainder of the study.

2. Analysis of Adriamycin-induced nephropathy in wild-type and MASP-2-/-mice

To elucidate the role of the lectin pathway of complement in doxorubicin-induced nephropathy, MASP-2-/-mice (BALB/c) groups were compared with wild-type mice (BALB/c) at the same dose of doxorubicin. MASP-2-/-mice were backcrossed to BALB/c mice for 10 passages.

Wild type (n=8) and MASP-2-/- (n=8) were IV injected with doxorubicin (10.5 mg/kg), and three mice of each strain were given saline alone as a control. All mice were sacrificed two weeks after treatment and tissues were collected. The extent of histopathological damage was assessed by H & E staining.

Results:

fig. 37 shows representative H & E stained tissue sections from the following groups of mice on day 14 after treatment with doxorubicin alone or saline (control): (panels A-1, A-2, A-3) wild-type control mice treated with saline alone; (panels B-1, B-2, B-3) wild-type mice treated with doxorubicin; and (panels C-1, C-2, C-3) MASP-2-/-mice treated with doxorubicin. Each photograph (e.g., panels A-1, A-2, A-3) represents a different mouse.

As shown in FIG. 37, there was a much higher degree of tissue preservation in the MASP-2-/-group treated with doxorubicin than in the wild-type group treated with the same dose of doxorubicin.

Figure 38 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with macrophage specific antibody F4/80, showing the average staining area (%) of macrophages from the following mice groups on day 14 after treatment with doxorubicin alone or saline (wild type control): wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2-/-mice treated with saline only, and MASP-2/-mice treated with doxorubicin. As shown in fig. 38, MASP-2-/-mice treated with doxorubicin had reduced macrophage infiltration compared to wild-type mice treated with doxorubicin (p=0.007).

Figure 39 graphically illustrates the results of computer-based image analysis of kidney tissue sections stained with sirius red, showing the areas of staining (%) of collagen deposition from the following groups of mice on day 14 after treatment with doxorubicin alone or saline (wild type control): wild-type control mice treated with saline alone; wild-type mice treated with doxorubicin; MASP-2-/-mice treated with saline only, and MASP-2/-mice treated with doxorubicin. As shown in fig. 39, MASP-2-/-mice treated with doxorubicin had reduced collagen deposition (×p=0.005) compared to wild-type mice treated with doxorubicin.

General overview and conclusion：

Improvement of tubular interstitial inflammation is a key target for kidney disease treatment. The results presented herein indicate that the complement-activated lectin pathway contributes significantly to the development of tubular interstitial inflammation. As further demonstrated herein, MASP-2 inhibitors, such as MASP-2 inhibitory antibodies, can be used as novel therapies for treating proteinuria nephropathy, doxorubicin nephropathy, and ameliorating tubular interstitial inflammation.

Example 19

This example describes preliminary results of an ongoing phase 2 clinical trial to assess the safety and clinical efficacy of fully human monoclonal MASP-2 inhibitory antibodies in adults with steroid-dependent immunoglobulin A nephropathy (IgAN), and in adults with steroid-dependent Membranous Nephropathy (MN).

Background：

Chronic kidney disease affects more than 2000 thousands of people in the united states (Drawz p. Et al, ann international Med 162 (11); ITC1-16, 2015). Glomerular kidney disease (GN), including IgAN and MN, is a kidney disease in which glomeruli are damaged and frequently results in end-stage kidney disease and dialysis. There are several types of primary GNs, most commonly igans. Many of these patients have persistent kidney inflammation and progressive deterioration. These patients are often treated with corticosteroids or immunosuppressants, which have many serious long-term adverse consequences. Even under these treatments, many patients continue to deteriorate. There is no approved treatment for treating IgAN or MN.

IgA nephropathy

Immunoglobulin a kidney disease (IgAN) is an autoimmune kidney disease, resulting in intrarenal inflammation and kidney injury. IgAN is the most common primary glomerular disease worldwide. With an annual incidence of about 2.5/100,000, 1 of 1400 us was estimated to develop IgAN. Up to 40% of IgAN patients develop end-stage renal disease (ESRD). Patients often present with microscopic hematuria with mild to moderate proteinuria and variable levels of renal insufficiency (Wyatt r.j. Et al, N Engl J Med 368 (25): 2402-14, 2013). Clinical markers such as impaired renal function, sustained hypertension and severe proteinuria (over 1 g/day) are associated with poor prognosis (Goto M et al, nephrol Dial Transplant (10): 3068-74, 2009; berthoux F. Et al, J Am Soc neprol 22 (4): 752-61, 2011). Proteinuria is the strongest prognostic factor in multiplex large-scale observational studies and prospective trials independent of other risk factors (Coppo R. Et al, JNaephrol 18 (5): 503-12, 2005; reich H.N. et al, J Am Soc Nephrol18 (12): 3177-83, 2007). If untreated, it is estimated that 15-20% of patients reach ESRD (D' Amico G., am J Kidney Dis 36 (2): 227-37, 2000) within 10 years of the onset of the disease.

Diagnostic markers for IgAN are IgA deposition in the glomerular mesangium, either alone or together with IgG, igM or both. In IgAN, kidney biopsies revealed glomerular deposition of mannan-binding lectin (MBL), a key recognition molecule for MASP-2 (effector enzyme of the lectin pathway of the complement system) activation. Glomerular MBL deposits that are commonly co-localized with IgA and indicate complement activation, as well as high levels of urine MBL are associated with adverse prognosis in IgAN, where these patients demonstrate more severe histological changes and mesangial hyperplasia than patients without MBL deposition or with high levels of urine MBL (Matsuda m. Et al, nepron 80 (4): 408-13, 1998; liu LL et al, clin Exp Immunol 169 (2): 148-155, 2012; roos a. Et al, J Am Soc neprol 17 (6): 1724-34, 2006; liu et al, clin Exp Immunol 174 (1): 152-60, 2013). The rate of remission is also substantially lower for patients with MBL deposition (Liu LL et al, clin Exp Immunol 174 (1): 152-60, 2013).

Current therapies for IgAN include blood pressure control, and frequently, corticosteroids and/or other immunosuppressants for severe diseases (e.g., crescent-shaped IgAN), such as cyclophosphamide, azathioprine, or mycophenolate. Kidney Disease Improving Global Outcomes (KDIGO) Guidelines for Glomerulonephritis (int. Soc of Nephrol2 (2): 139-274, 2012), recommends that corticosteroids should be administered to patients with proteinuria of greater than or equal to 1 g/day for a typical treatment duration of 6 months. However, even with invasive immunosuppressive therapy (which is associated with serious long-term sequelae), some patients have a progressive deterioration of renal function. There is no approved treatment for IgAN and even with Angiotensin Converting Enzyme (ACE) inhibitors or Angiotensin Receptor Blockers (ARBs) to control blood pressure, increased proteinuria persists in some patients. None of these treatments has been shown to stop or even slow the progression of IgAN in patients at risk of rapid disease progression.

Membranous nephropathy

The annual incidence of Membranous Nephropathy (MN) is about 10-12/1,000,000.MN patients may have variable clinical course, but approximately 25% develop end-stage renal disease.

Membranous nephropathy is one of the most common causes of immune-mediated glomerular disease, as well as nephrotic syndrome in adults. The disease is characterized by the formation of immune deposits (mainly IgG 4) on the outside of glomerular basement membrane, which contain podocyte antigens and antibodies specific for those antigens, resulting in complement activation. The initial manifestation of MN is associated with nephrotic syndrome: proteinuria, hypoalbuminemia, hyperlipidemia and oedema.

Although MN may spontaneously alleviate without treatment, up to one third of patients demonstrate a gradual loss of kidney function and progression to ESRD at the median 5 years after diagnosis. Corticosteroids are often used to treat MN and alternative therapies need to be developed. In addition, based on the severity of proteinuria, patients determined to be at moderate risk of progression are treated with prednisone in combination with cyclophosphamide or calmodulin inhibitors, and both treatments are often associated with serious systemic adverse effects.

Method：

Two phase 1 clinical trials in healthy volunteers have demonstrated that both intravenous and subcutaneous administration of MASP-2 inhibitory antibody OMS646 resulted in sustained lectin pathway inhibition.

This example describes the interim results of an ongoing phase 2, non-control, multicenter study of the MASP-2 inhibitory antibody OMS646 in subjects with IgAN and MN. Inclusion criteria require that all patients in this study, regardless of the renal disease subtype, have been maintained at a stable dose of corticosteroid for at least 12 weeks prior to study enrollment (i.e., the patients are steroid dependent). The study was a single group (arm) pilot study with a 12 week treatment period and a 6 week follow-up period.

Approximately four subjects/diseases are planned to enroll. The study was designed to assess whether OMS646 can improve kidney function (e.g., improve proteinuria) and reduce corticosteroid requirements in both IgAN and MN subjects. To date, 2 IgA nephropathy patients and 2 membranous nephropathy patients have completed the treatment in this study.

At study entry, each subject must have high levels of protein in the urine despite continued treatment with a stable corticosteroid dose. These criteria selected patients who were unlikely to spontaneously improve during the study period.

Subjects were ≡18 years old at screening and were included in the study only when they had one of the following diagnoses: igAN diagnosed at renal biopsy or primary MN diagnosed at renal biopsy. Enrolled patients must also meet all of the following inclusion criteria:

(1) Prior to each of the 2 visits during the screening period, there was an average urinary albumin/creatinine ratio >0.6 from three samples collected continuously and daily.

(2) At least 12 weeks prior to screening visit 1 at a dose of ≡10mg prednisone or equivalent;

(3) If under immunosuppressive therapy (e.g., cyclophosphamide, mycophenolate mofetil), then at least 2 months had been at a steady dose prior to screening visit 1, there was no predicted dose change for the duration of the study;

(4) With passage through the MDRD equation ¹ Calculated estimated glomerular filtration rate (eGFR). Gtoreq.30 mL/min/1.73 m ² ；

(5) A stable, optimized treatment with an Angiotensin Converting Enzyme Inhibitor (ACEI) and/or an Angiotensin Receptor Blocker (ARB) at the direction of a physician and having a systolic blood pressure of <150mmHg and a diastolic blood pressure of <90mmHg at rest;

(6) No belimumab, eculizumab or rituximab was used within 6 months of screening visit 1; and

(7) There was no history of kidney transplantation.

¹ MDRD equation: eGFR (mL/min/1.73 m) ² )＝175x(SCr) ^-1.154 x (age) ^-0.203 x (0.742 in case of female) x (1.212 in case of african americans). Note that: SCr = serum creatinine measurement should be mg/dL.

The monoclonal antibody OMS646 used in this study was a fully human IgG4 monoclonal antibody that bound to and inhibited human MASP-2. MASP-2 is an effector enzyme of the lectin pathway. As demonstrated in example 12, OMS646 binds affinity to recombinant MASP-2 (apparent equilibrium dissociation constant in the range of 100 pM) and shows greater than 5,000-fold selectivity over homologous proteins C1, C1r and MASP-1. In a functional assay, OMS646 was shown to be nanomolar in potency (resulting in about 3nM 50% inhibition [ IC ₅₀ ]Is present) inhibits the human lectin pathway but has no significant effect on the classical pathway. OMS646 administered by Intravenous (IV) or Subcutaneous (SC) injection to mice, non-human primates and humans resulted in high plasma concentrations, which are associated with suppression of lectin pathway activation in ex vivo assays.

In this study, OMS646 drug substance was provided at a concentration of 100mg/mL, which was further diluted for IV administration. An appropriate calculated volume of OMS646 100mg/mL injection solution was drawn from the vial using a syringe for dose preparation. The infusion bag was administered within four hours after preparation.

The study consisted of screening (28 days), treatment (12 weeks) and follow-up (6 weeks) periods, as shown in the study design schematic below.

Schematic of research design

During the screening period and prior to the first OMS646 dose, the consented subjects provided three urine samples (collected once a day) each over two consecutive three-day periods to establish a baseline value for urinary albumin/creatinine ratio. After the screening period, eligible subjects received OMS646 4mg/kg IV once a week for 12 weeks (treatment period). There was a 6 week follow-up period following the last dose of OMS 646.

During the first 4 weeks of treatment with OMS646, subjects were maintained at their stable pre-study corticosteroid dose. At the end of the first 4 weeks of the 12-week treatment period, the subject experienced a gradual decrease in corticosteroid (i.e., a decrease in corticosteroid dose), if tolerated, for 4 weeks, followed by 4 weeks during which the resulting corticosteroid dose was maintained. The goal is to gradually decrease to less than or equal to 6mg of prednisone (or equivalent dose) per day. Over this period, as determined by the investigator, the gradual decrease ceased in subjects with worsening renal function. Subjects were treated with OMS646 by treatment with corticosteroid gradually decreased and throughout 12 weeks. The patient was then followed up for another 6 weeks after its last treatment. The gradual decrease in corticosteroid and OMS646 treatment allows for evaluation of whether OMS646 allows for a decrease in the corticosteroid dose required to maintain stable kidney function.

The key efficacy measures in this study were the change from baseline to 12 weeks, urinary albumin/creatinine ratio (uACR) and 24 hour protein levels. Measurement of urine proteins or albumin is routinely used to assess renal involvement, and sustained high levels of urine proteins are associated with renal disease progression. uACR is used clinically to evaluate proteinuria.

Efficacy analysis

The analytical value of uACR is defined as the average of all values obtained for a certain point in time. The number of plans for the uACR is three at each planned time point. The baseline value of uACR is defined as the mean of the analysis values at the time of two screening visits.

Results:

FIG. 40 graphically illustrates uACR in two IgAN patients during the course of twelve weeks of weekly studies with 4mg/kg MASP-2 inhibitory antibody (OMS 646). As shown in fig. 40, the change from baseline was analyzed by unconverted, at time point "a" (p=0.003); is statistically significant at time point "b" (p=0.007) and time point "c" (p=0.033). Table 12 provides 24 hour urine protein data for two IgAN patients treated with OMS 646.

Table 12: 24 hour urine protein (mg/day) in OMS646 treated IgAN patients

As shown in fig. 40 and table 12, the IgAN patients demonstrated clinically and statistically significant improvement in kidney function over the course of the study. There was a statistically significant decrease in both uACR (see fig. 40) and 24 hour urine protein concentration (see table 12). As shown in the uACR data in fig. 40, the average baseline uACR was 1264mg/g and reached 525mg/g at the end of the treatment (p=0.011), decreasing to 128mg/g at the end of the follow-up period. As further shown in fig. 40, the therapeutic effect was maintained throughout the follow-up period. Measurement of 24 hours urine protein excretion followed the uACR with an average decrease from 3156mg/24 hours to 1119mg/24 hours (p=0.017). The therapeutic effect across two patients is highly consistent. Both patients experienced a decrease of about 2000 mg/day and both achieved partial remission (defined as greater than 50 percent decrease in urine protein excretion at 24 hours and/or less than 1000 mg/day resulting protein excretion; complete remission defined as less than 300 mg/day protein excretion). The magnitude of 24-hour proteinuria reduction in both IgA nephropathy patients was associated with a significant improvement in kidney survival. Two IgA nephropathy patients were also able to substantially gradually reduce their steroid dose, each reducing the daily dose to 5mg (60 mg to 0mg;30mg to 5 mg).

Two MN patients also demonstrated a decrease in uACR during treatment with OMS 646. One MN patient had a decrease in uACR from 1003mg/g to 69mg/g and was maintained at this low level throughout the follow-up period. Another MN patient had a decrease in uACR from 1323mg/g to 673mg/g with variable course after treatment. The first MN patient showed a significant decrease in urine protein levels for 24 hours (10,771 mg/24 hours at baseline to 325mg/24 hours at day 85) with partial and almost complete remission being achieved, while the other patient remained essentially unchanged (4272 mg/24 hours at baseline to 4502mg/24 at day 85). The steroid was gradually reduced in both MN patients, from 30mg to 15mg, and from 10mg to 5mg.

In summary, consistent improvement in renal function was observed in IgAN and MN subjects treated with MASP-2 inhibitory antibody OMS 646. The effect of OMS646 treatment in IgAN patients was robust and consistent suggesting a strong efficacy signal. These effects are supported by results in MN patients. The time course and magnitude of the uACR changes during treatment were consistent in all four IgAN and MN patients. No significant safety issues were observed. Patients in this study represent a difficult treatment group, and the efficacy in these patients is considered to predict efficacy with MASP-2 inhibitory antibodies, such as OMS646, in IgAN and MN patients, e.g., patients with steroid-dependent IgAN and MN (i.e., patients undergoing treatment with a stable corticosteroid dose prior to treatment with MASP-2 inhibitory antibodies), including patients at risk of rapidly progressing to end-stage renal disease.

In accordance with the foregoing, in one embodiment, the present invention provides a method of treating a human subject having IgAN or MN comprising administering to the subject a composition comprising an amount of MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the method comprises administering an amount of a MASP-2 inhibitory antibody sufficient to improve kidney function (e.g., improve proteinuria) to a human subject having IgAN or MN. In one embodiment, the subject has steroid dependent IgAN. In one embodiment, the subject has a steroid dependent MN. In one embodiment, the MASP-2 inhibitory antibody is administered to a subject suffering from a steroid dependent IgAN or steroid dependent MN in an amount sufficient to improve kidney function and/or reduce corticosteroid dosage in said subject.

In one embodiment, the method further comprises identifying a human subject having steroid dependent IgAN prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation.

In one embodiment, the method further comprises identifying a human subject having a steroid dependent MN prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation.

According to any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10nM or less, the antibody binds to an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum with an IC50 of 10nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, fab, fab ', F (ab) 2, and F (ab') 2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway, and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complement pathway intact).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with renal function, such as improvement in proteinuria (e.g., a decrease in uACR and/or a decrease in 24-hour urine protein concentration, such as a greater than 20 percent decrease in 24-hour urine protein excretion, or a greater than 30 percent decrease in 24-hour urine protein excretion, or a greater than 40 percent decrease in 24-hour urine protein excretion, or a greater than 50 percent decrease in 24-hour urine protein excretion, for example).

In some embodiments, the method comprises administering the MASP-2 inhibitory antibody to a subject having IgAN (e.g., steroid-dependent IgAN) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to one week or two weeks or three weeks or four weeks or more), followed by subcutaneously administering the MASP-2 inhibitory antibody to the subject for a second period of time (e.g., a chronic period of at least two weeks or more).

In some embodiments, the method comprises administering the MASP-2 inhibitory agent to a subject having MN (e.g., steroid dependent MN) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to one week or two weeks or three weeks or four weeks or more), followed by subcutaneously administering the MASP-2 inhibitory antibody to the subject for a second period of time (e.g., a chronic period of at least two weeks or more).

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody intravenously, intramuscularly, or subcutaneously to a subject having IgAN (e.g., steroid-dependent IgAN) or MN (e.g., steroid-dependent MN). The treatment may be chronic and is administered daily to monthly, but is preferably at least once every two weeks or weekly, for example twice weekly or three times weekly.

In one embodiment, the method comprises treating a subject having IgAN (e.g., steroid dependent IgAN) or MN (e.g., steroid dependent MN) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence as set forth in SEQ ID NO:67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence as set forth in SEQ ID NO: 69. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising: (a) a heavy chain variable region comprising: i) A heavy chain CDR-H1 comprising an amino acid sequence from 31 to 35 of SEQ ID NO. 67; and ii) a heavy chain CDR-H2 comprising an amino acid sequence from 50 to 65 of SEQ ID NO. 67; and iii) a heavy chain CDR-H3 comprising an amino acid sequence from 95 to 107 of SEQ ID NO. 67, and b) a light chain variable region comprising: i) Light chain CDR-L1 comprising the amino acid sequence of 24-34 from SEQ ID NO. 69; and ii) light chain CDR-L2 comprising the amino acid sequence from 50 to 56 of SEQ ID NO. 69; and iii) a light chain CDR-L3 comprising an amino acid sequence from 89-97 of SEQ ID NO. 69, or (II) a variant thereof comprising a heavy chain variable region having 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO. 67, and a light chain variable region having 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO. 69.

In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 that is recognized by reference antibody OMS646, which reference antibody OMS646 comprises a heavy chain variable region as set forth in SEQ ID NO:67 and a light chain variable region as set forth in SEQ ID NO: 69.

In some embodiments, the method comprises administering to a subject having or at risk of developing IgAN (e.g., steroid dependent IgAN) or MN (e.g., steroid dependent MN) a composition comprising a MASP-2 inhibitory antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:69, at a dose of 1mg/kg to 10mg/kg (i.e., 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, or 10 mg/kg), at least once per week (e.g., at least twice per week or at least three times per week), for at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 9 weeks, at least 10 weeks, at least 11 weeks or at least 12 weeks.

Example 20

This example describes a study using the MASP-2 inhibitory antibody OMS646 in the treatment of subjects suffering from or at risk of developing coronavirus-induced acute respiratory distress syndrome.

Background/principle：

Acute respiratory distress syndrome is a serious complication of coronavirus infection. SARS-CoV occurs in circulating coronaviruses on the animal market, resulting in global outbreaks of respiratory disease, with more than 8,000 individual cases and a mortality rate of 10% (Rota P.A. et al, science300:1394-1999, 2003). In 2012, a new related coronavirus was identified in the middle east, designated as the middle east respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease with a mortality rate of greater than 35% (Zaki a.m. et al, N Engl J Med 367:1814-1820, 2012). Coronavirus disease 2019 (covd-19) is an infectious disease that occurs in 2019 and is caused by severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2 or SARS-CoV-2), a virus closely related to the SARS virus (world health organization, 2/11/2020,Novel Coronavirus Situation Report 22). Covd-19, SARS-CoV and MERS-CoV all cause a range of diseases ranging from asymptomatic cases to severe acute respiratory distress syndrome (coronavirus induced ARDS) and respiratory failure. Those affected by covd-19 may develop fever, dry cough, fatigue, and shortness of breath. Findings on computed tomography can reveal lung ground glass shadows and double-sided flocculent shadows. Cases may progress to respiratory dysfunction, including pneumonia, severe acute respiratory distress syndrome, which can lead to multiple organ failure and septic shock, as well as death in the weakest person (see, e.g., hui d.s. Et al, int J effect Dis 91:264-266, month 14 of 2020, and et al OI:10.1056/NEJMoa 2002032). There is no vaccine or specific antiviral therapy, which manages treatments involving symptoms and supportive care. Thus, there is an urgent need to develop therapeutically effective agents to treat, inhibit and/or prevent coronavirus-induced acute respiratory distress syndrome.

Complement activation has been observed to contribute to the pathogenesis of coronavirus-induced severe acute respiratory syndrome. Mice infected with SARS-CoV that were deficient in complement component 3 (C3-/-mice) were found to exhibit significantly less weight loss and less respiratory dysfunction than C57BL/6J control mice infected with SARS-CoV, despite the equivalent viral load in the lung (Gralinski L.E. et al, mBio9:e01753-18, 2018). It was further observed that significantly fewer neutrophils and inflammatory monocytes were present in the lungs of the SARS-CoV-infected C3-/-mice compared to the infected control mice, and that in the lungs of the SARS-CoV-infected C3-/-mice, reduced pulmonary pathology and lower cytokine and chemokine levels (e.g., IL-5, IL-6) compared to the infected control mice (Gralinski L.E. et al, mBio9: e01753-18, 2018).

Studies have also shown that many survivors of SARS-CoV infection develop pulmonary fibrosis with a higher prevalence in elderly patients (Hui d.s. et al, chest 128:2247-2261, 2005). There are limited options for treating pulmonary fibrosis, such as coronavirus-induced fibrosis. Traditionally, corticosteroids are used to treat ARDS and pulmonary fibrosis, however, during viral infection, this treatment impairs the immune response and can lead to disease progression (Gross t.j. Et al, N Engl J Med 345:517-525, 2001).

As noted previously, there is no effective treatment for covd-19 and the disease is in rapid transmission. Although mortality assessment is still early, the world health organization reports a mortality rate of 3.4% in the last 3 rd month of 2020 (world wide web. Who.int/dg/speches/distail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19- -3 rd month of 2020). There is a need for effective treatment for patients with severe covd-19 infection.

As described herein, the lectin pathway is one of three activation pathways for complement. Other pathways are classical and alternative. All activation pathways lead to the production of the anaphylatoxins C3a and C5a, and the production of C5b-9 or Membrane Attack Complexes (MACs) on target cells.

The lectin pathway is part of the innate immune system and is activated by microorganisms or damaged cells. Microorganisms display carbohydrate-based pathogen-associated molecular patterns (PAMPs), and damaged host cells display damage-associated molecular patterns (DAMP). DAMPs are not displayed on healthy cells, but become exposed as cells become damaged.

Circulating lectins, such as Mannose Binding Lectin (MBL), fiber gelation proteins, and collectins recognize and bind PAMPs and DAMP. Lectins that bind to PAMPs or DAMP localize complement activation in the vicinity of the cell membrane. These lectins carry mannans that bind lectin associated serine protease 2 (MASP-2), which cleaves complement factors 2 and 4 to produce C3 convertases, which in turn cleave C3 to form C5 convertases. In addition to lectin pathway activation, alternative pathways can also be activated and complement activation expanded. All of this results in the insertion of MACs into the membrane of damaged cells, causing further damage to the cells, with more DAMPs exposed. The circulating lectin carrying MASP-2 recognizes and binds to DAMP, resulting in further lectin pathway activation and additional cell damage. In this way, the lectin pathway can amplify and exacerbate cellular damage caused by initial complement activation.

As described herein, OMS646 (also referred to as OMS721 or nano-cord Li Shan antibodies) is a research human IgG4 monoclonal antibody directed against MASP-2. Activation of the lectin pathway is inhibited by blocking MASP-2, as further described herein. This may disrupt the complement-mediated cell damage cycle described above. To date, OMS646 has been administered to approximately 230 healthy volunteers, thrombotic Microangiopathy (TMA) patients, and glomerulonephropathy such as immunoglobulin a [ IgA ] nephropathy patients.

The lectin pathway may be initiated in coronavirus-induced ARDS and play a key role in sustained complement activation, and inhibition via the lectin pathway of MASP-2 inhibitors (e.g., MASP-2 inhibitory antibody OMS 646) may address complement-mediated lung injury associated with coronavirus infection. As described herein, the inventors have discovered that inhibition of the key regulator of the lectin pathway of the complement system, namely mannan-binding lectin associated serine protease 2 (MASP-2), significantly reduces inflammation and fibrosis in various animal models of fibrotic disease. For example, the results presented in examples 14 and 15 herein demonstrate the beneficial effects of MASP-2 inhibition on tubular interstitial inflammation, tubular cell injury, pro-fibrotic cytokine release and scarring. As described in example 17, in an analysis of monoclonal MASP-2 inhibitory antibodies with respect to efficacy in reducing and/or preventing renal inflammation and tubular interstitial damage in a mouse protein overload proteinuria model in wild-type mice, a significant reduction in IL-6 levels in the group in which MASP-2 inhibitory antibody treatment was present was determined as compared to the saline control group (p=0.0269) and the isotype control group (p=0.0445), as shown in fig. 36.

The method comprises the following steps:

the following study was conducted to analyze the use of OMS646 in treating one or more patients suffering from a coronavirus (e.g., covd-19-virus) infection in order to measure the efficacy of OMS646 in treating, inhibiting, reducing or preventing acute respiratory distress syndrome in the patient.

The method involves identifying a subject infected with a coronavirus, such as covd-19, MERS-CoV, or SARS, which can be determined by conducting a diagnostic test, such as a molecular test (e.g., rRT-PCR) or serological test, or referencing a database containing such information. Exemplary tests for COVID-19, MERS-CoV and SARS are found on the disease control center (Centers For Disease Control) website (world-wide-web. Cdc.gov/corenavirus/MERS/lab/lab-testing. Html#molecular).

The subject may have, or be at risk of developing, a covd-19 induced ARDS, e.g., a subject with pneumonia. Pneumonia is the most common risk factor for ARDS development (Sweeney r.m. and McAuley, d.f., lancet volume 388:2416-30, 2016).

Covd-19 induced ARDS is defined as a clinical syndrome that develops after infection by covd-19 and meets one or more of the following ARDS criteria (Sweeney r.m. and McAuley, d.f., lancet volume 388:2416-30, 2016), based on the berlin definition (JAMA 307:2526, 2012):

Oxygenation (mm Hg): light (PaO) ₂ /FiO ₂ 200-300); moderate (PaO) ₂ /Fi O ₂ 100-199); severe (PaO) ₂ /FiO ₂ <100)

Positive End Expiratory Pressure (PEEP) (cm H) ₂ O): the minimum PEEP required is 5

Infiltration on chest radiographs: bilateral infiltration involving two or more quadrants on frontal She Shexian photographs or CT

Heart failure: left heart failure alone, which is not sufficiently responsible for clinical status

Severity: based on oxygenation criteria

Therapeutic administration

A subject suffering from COVID-19 and experiencing one or more respiratory symptoms (such as those criteria set forth above) is administered 4mg/kg OMS646 via intravenous infusion. Treatment was administered twice weekly. The frequency of doses is guided by the patient response to therapy. If the patient demonstrates a clinical improvement for 4 weeks, the dose can be reduced to 4mg/kg once a week. If the patient maintains the therapeutic response for 4 weeks at 4mg/kg once a week, treatment may be discontinued.

Positive response to treatment is determined when an improvement is observed in respiratory function, such as one or more respiratory symptoms, such as one or more ARDS criteria.

In accordance with the foregoing, in one aspect, the present invention provides a method for treating, inhibiting, reducing or preventing acute respiratory distress syndrome or other manifestations of a disease in a mammalian subject infected with a coronavirus comprising administering to the subject an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation (i.e., inhibit lectin pathway activation). In some embodiments, the subject suffers from one or more respiratory symptoms, and the method comprises administering to the subject an amount of a MASP-2 inhibitor effective to improve MASP-2 dependent complement activation (i.e., improve respiratory function).

In one embodiment, the method comprises administering the composition to a subject infected with covd-19. In one embodiment, the method comprises administering the composition to a subject infected with SARS-CoV. In one embodiment, the method comprises administering the composition to a subject infected with MERS-CoV. In one embodiment, the subject is identified as having a coronavirus (i.e., covd-19, SARS-CoV, or MERS-CoV) prior to administration of the MASP-2 inhibitor.

In one embodiment, the MASP-2 inhibitor is a small molecule that inhibits MASP-2 dependent complement activation.

In one embodiment, the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% of human serum, IC thereof ₅₀ 30nM ofOr smaller.

In one embodiment, a MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence as set forth in SEQ ID NO. 67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence as set forth in SEQ ID NO. 69. In one embodiment, a MASP-2 inhibitory antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69.

In some embodiments, the method comprises administering to a subject infected with a coronavirus a composition comprising a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, that comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID No. 67, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID No. 69, at a dose of 1mg/kg to 10mg/kg (i.e., 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, or 10 mg/kg), at least once per week (e.g., at least twice per week or at least three times per week), for a period of at least 2 weeks (e.g., at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 9 weeks, or at least 10 weeks, or at least 11 weeks, or at least 12 weeks).

In one embodiment, the dosage of MASP-2 inhibitory antibody is about 4mg/kg (i.e., 3.6mg/kg to 4.4 mg/kg).

In one embodiment, the dose of MASP-2 inhibitory antibody is a fixed dose of about 300mg to about 450mg (i.e., about 300mg to about 400mg, or about 350mg to about 400 mg), such as about 300mg, about 305mg, about 310mg, about 315mg, about 320mg, about 325mg, about 330mg, about 335mg, about 340mg, about 345mg, about 350mg, about 355mg, about 360mg, about 365mg, about 370mg, about 375mg, about 380mg, about 385mg, about 390mg, about 395mg, about 400mg, about 405mg, about 410mg, about 415mg, about 420mg, about 425mg, about 430mg, about 435mg, about 440mg, about 445mg, and about 450 mg. In one embodiment, the dose of MASP-2 inhibitory antibody is a fixed dose of about 370mg (+ -10%).

In one embodiment, the method comprises administering intravenously a fixed dose of about 370mg (+ -10%) of MASP-2 inhibitory antibody to a subject infected with coronavirus twice weekly for a treatment period of at least 8 weeks.

In one embodiment, the MASP-2 inhibitor is delivered systemically to the subject. In one embodiment, the MASP-2 inhibitor is administered orally, subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, or as an inhalant.

Example 21

OMS646 (Naxol Li Shan antibody) treatment in COVID-19 patients

This example describes the use of naloxone Li Shan anti (OMS 646) in the treatment of a patient infected with covd-19 using the method described in example 20. The results described in this example confirm the efficacy of naloxone Li Shan against the patient with covd-19 described in example 20.

Background/principle:

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19) was identified as a clinical syndrome and transmitted rapidly. By late 2 months in 2020, a rapidly growing number of cases of COVID-19 were diagnosed in Lombardy region in North Italy (Remuzzi A, remuzzi G. COVID-19and Italy:what nextThe Lancet). The main cause of death for covd-19 is severe respiratory dysfunction. Lung tissue in patients who have died from covd-19 shows high concentrations of SARS-CoV RNA (Wichmann d. Et al, ann international Med, 2020), and the same intensity of inflammatory changes are seen in previously reported coronaviruses SARS-CoV (SARS) and MERS-CoV (MERS), and anti-inflammatory strategies are being evaluated for covd-19 treatment (Xu z. Et al, lancet Respir Med,8 (4): 420-2, 2020; horby P. Et al, midrxiv 2020:2020.06.22.20137273;Gritti G. Et al, midrxiv 2020.04.01.20048561). Thrombosis has also been reported in SARS and COVID-19 infection (Wichmann D. Et al, ann International Med 2020; magro C. Et al, transl Res 2020; ding Y. Et al, J Pathol 200 (3): 28209, 2003). Like SARS and MERS, covd-19 can cause life threatening Acute Respiratory Distress Syndrome (ARDS) (Guan W.J, ni Z.Y, hu Y et al Clinical Characteristics of Coronavirus Disease 2019in China.N Engl J Med 2020[ early online publication ]).

The key pathological components of the COVID-19 and ARDS exudation phases are endothelial injury and activation (Varga Z. Et al, lancet 2020; ackermann M. Et al, N Engl J Med 2020; green S.J. Et al, microbes select 22 (4-5): 149-50, 2020; teuwen L.A. Et al, nat Rev Immunol 20 (7): 389-91, 2020; goshua G. Et al, lancet Haemato 2020;Thompson B.T. Et al, N Engl J Med 377 (19): 1904-5, 2017). The increased capillary permeability and root cause of pulmonary edema in ARDS, endothelial injury can also cause microvascular vascular disease and thrombosis. Endothelial injury can also cause microvascular vascular disease and thrombosis. Endothelial activation further enhances the local inflammatory environment. Importantly, as demonstrated in vitro and animal studies, endothelial injury specifically activates the lectin pathway of complement on the surface of endothelial cells (cold CD,a, morrissey MA et al Complement Activation after Oxidative Stress: role of the Lectin Complement Pathway.am J Pathol 2000;156 (5):1549-1556).

OMS646 (also known as Naxo Li Shan antibody) was predicted to be effective in treating patients with COVID-19 as described in example 20, a high affinity monoclonal antibody that binds MSP-2 and blocks lectin pathway activation. Consistent with the description in example 20, MASP-2 has been directly linked to lung injury in coronavirus infection in animal models. See Gao et al, medRxiv 3/30/2020.MASP-2 also acts directly on the coagulation cascade and contact system, cleaving prothrombin into thrombin and forming a fibrin clot. The nano-cord Li Shan inhibits not only lectin pathway activation, but also blocks microvascular injury-related thrombosis, as well as MASP-2 mediated activation of kallikrein and factor XII.

No disease-specific therapies have been shown to be effective in treating covd-19. In view of the heavy disease burden of italy, we treated patients with severe covd-19 infection and ARDS with the naso Li Shan antibody under the contemporaneous use plan at Papa Giovanni XXIII Hospital of Bergamo. This represents the first time lectin pathway inhibitors have been used to treat patients with covd-19. Here we report this preliminary clinical experience.

Method

Study supervision

The investigation described in this example was performed in the Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII of Bergamo, italy, and was approved by the institutional ethics committee and Agenzia Italiana del Farmaco. Laboratory values were collected according to standard clinical practice, including blood count, LDH, C-reactive protein (CRP). All patients treated with naloxone Li Shan anti (OMS 646) provided informed consent. This study was performed using the method described in example 20, as described further below.

Histopathology

Standard hematoxylin and eosin staining (H & E) and immunohistochemistry were performed on formalin fixed, paraffin embedded samples from pathological necropsy of covd-19 patients. H & E stained sections were examined by two pathologists. To confirm the diagnosis, immunohistochemical analysis of human endothelial cell markers (CD 34) has been performed with Bond Ready-to-Use anti-body CD34 (Clone QBEnd/10,Leica Biosystems, germany) using a specifically optimized Ready-to-Use product for Bond Polymer Refine Detection. The assay was performed on an automated stainer platform (Leica Bond-3, leica, germany) using a heat-based antigen retrieval technique (Bond Epitope Retrieval solution 2 for 20 minutes) as recommended by the manufacturer. Cytoplasmic staining of the endothelium in alveolar capillaries indicated a positive result.

Identification and enumeration of Circulating Endothelial Cells (CECs)

CECs were tested by flow cytometry analysis performed on peripheral blood samples collected with EDTA. After the erythrocyte lysis step, the samples were labeled with the following monoclonal antibodies for 20 minutes at room temperature:anti-CD 45V 500 (clone 2D1,Becton Dickinson,San Jose ', CA), anti-CD 34 PerCP-CY5.5 (clone 8G12,Becton Dickinson,San Jose', CA), anti-CD 146 PE (clone P1H12BD, pharmingen, CA). At least 1×10 with total leukocyte morphology was obtained by flow cytometry (FACSLyric, BD Biosciences) ⁶ Event/sample. To reduce operator induced variability, all samples in this study were analyzed by the same laboratory technician at all times. As previously reported, CEC/ml numbers were calculated by a double platform counting method using lymphocyte subsets as reference populations (almini c. Et al Bone Marrow Transplant 52:1637-42, 2017).

Serum levels of cytokines

The levels of interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor Necrosis Factor (TNF) and interleukin 12p70 (IL-12 p 70) were analyzed by flow cytometry (BD CBA Human Inflammatory Cytokines Kit, becton Dickinson, san Jose, calif.) in a single serum sample.

Patient(s)

Between 11 and 23 days 3 and 3 months 2020, all patients resistant to the treatment with nano-cable Li Shan were admitted. Over this 13 day span, the total daily number of covd-19 patients admitted to the ward ranged from 405 to 542. During this same period, 140 helmet Continuous Positive Airway Pressure (CPAP) devices were utilized on average daily, and the ICU managed the median of 82 patients per day (range 66-91). Among these ICU patients, 61 on 11 months 3 and 80 on 23 months 3 in 2020 met the ARDS' S Berlin standard (PaO 2/FiO2 ratio <100 is severe ARDS;100-200 is moderate; >200 and +.ltoreq.300 is mild) (Ferguson N.D. et al Intensive Care Med (10): 1573-82, 2012; fagiuoli S. Et al, N Engl J Med 382 (21) e71, 2020).

All patients treated in this study had laboratory-confirmed infection with covd-19 diagnosed by quantitative reverse transcriptase-polymerase chain reaction assay. Detection of SARS-CoV-2 Gene from nasal and respiratory samples by different molecular methodsGroup, the molecular method includes GeneFinder ^TM Covid-19 Plus RealAmp Kit (elichech Group,92800Puteaux, france), allplexTM2019-nCoV Assay (Seegene Inc, arrow Diagnostics s.r.l., italy). After purification of viral RNA from clinical samples, detection of RdRp, E and N viral genes was obtained by real-time polymerase chain reaction according to the world health organization protocol (Corman V.M. et al, euro maintenance 25, 2020). In order to qualify for anti-treatment with Naso Li Shan, the patient who requires confirmation of covd-19 is adult >18 years old) and suffers from ARDS (Ferguson ND et al, responsive Care 38 (10): 1573-1582, 2012; see also Sweeney R.M. and McAuley, D.F., lancet volume 388:2416-30, 2016; JAMA 307:2526, 2012) and requires non-invasive mechanical ventilation by Continuous Positive Airway Pressure (CPAP) according to institutional guidelines for respiratory support. While all enrolled patients received 500mg of azithromycin empirically once a day, patients with active systemic bacterial or fungal infections requiring antimicrobial treatment were not eligible for halyard Li Shan anti-treatment.

Nasoh Li Shan anti-treatment, supportive therapy and outcome assessment

As described in example 20, the nano-cord Li Shan anti (OMS 646) is a fully human monoclonal antibody consisting of immunoglobulin gamma 4 (IgG 4) heavy and lambda light chain constant regions. It binds with sub-nanomolar affinity and inhibits MASP-2. According to the method described in example 20, naloxone Li Shan anti-tumor is administered intravenously twice weekly at a dose of 4mg/kg to 6 patients infected with covd-19 for 2 to 4 weeks, up to 6 to 8 doses (two weeks, three weeks or four weeks total). At study initiation, the duration of dosing was set to 2 weeks, but when the first patient treated with the naloxone Li Shan anti-treatment experienced clinical and laboratory marker recurrence after cessation of treatment at week 2, the duration of dosing was empirically increased, followed by regression with another week of dosing. At the time of the study, all patients received standard supportive care, including preventive enoxaparin (Clexane, sanofi Aventis) 4,000IU/0 4ml, azithromycin (Zitromax, pfizer SpA, italy) 500mg once a day, hydroxychloroquine (Plaquenil, sanofi Aventis) 200mg twice a day, darunavir and cobalastat (Rezolsta, janssen-Cilag s.p.a., italy) 800/150mg once a day, according to the guidelines of the hospital. Starting at day 27 of 3 months, all patients in the hospital who received COVID-19 received 1mg/kg of methylprednisolone according to the latest institutional guidelines. Thus, a total of 5 out of 6 patients treated with the naloxone Li Shan anti-treatment received also systemic corticosteroid (methylprednisolone 1 mg/kg) after initiation of the naloxone Li Shan anti-treatment. All respiratory support is provided according to institutional treatment algorithms. The clinical characteristics of these 6 patients are summarized in table 13 below.

In addition to CEC counts and cytokine levels, clinical and laboratory measurements including blood count, LDH and C-reactive protein (CRP) levels were collected for all patients resistant to treatment with nano-cable Li Shan according to standard clinical practice. Conventional blood tests were collected prior to each of the nano-cord Li Shan antibody doses and then twice weekly. Respiratory function was assessed daily. A chest Computed Tomography (CT) scan was performed on all patients at admission to record typical interstitial pneumonia and, if indicated clinically, pulmonary embolism. During the course of treatment, chest radiography is performed as clinically required.

Statistical analysis

Demographic and clinical patient data are presented as frequencies with percentages of the classification variable, with median values for ranges of continuous variables. The CEC value differences between normal and COVID-19 patients were assessed by the Mannheim U test. Repeated measurement assays were performed at appropriate time points to test differences in CEC and cytokine levels during the anti-treatment of nano-cable Li Shan; pairing comparisons were performed using a nonparametric friedemann test and using a paired wilcoxon symbol rank test. Between observation and time, the decreasing trend of LDH and CRP levels during treatment was assessed with a nonparametric spearman test. Significance was fixed at 5%. Analysis was performed using R software (version 3.6.2).

Table 13 summarizes the clinical characteristics of 6 patients treated with naloxone Li Shan.

Table 13: demographics of covd-19 patients treated with naloxone Li Shan

ARDS: acute respiratory distress syndrome; ICU: an intensive care unit; CPAP (continuous treatment): continuous positive airway pressure.

* : data available only for 4 patients

* *: data available only for 5 patients

# several patients were initially classified as having diabetes, but later reclassified as overweight but not having diabetes.

# is defined as body temperature >37.5 DEG C

Results:

thrombosis and endothelial cell damage in covd-19 patients

From day 3 month 13 to day 3 month 16, the hospital's pathology department began performing necropsy on an initial group of 20 deceased patients shortly after the onset of the noted covd-19 outbreak in the Bergamo region. All patients required advanced respiratory support with CPAP or invasive mechanical ventilation before their death, as with the patient currently being treated with naloxone Li Shan resistance in the study. Consistent with the clinical manifestations of frequently fatal pulmonary thromboembolism, the lungs and liver of many patients were found to be widely affected by thrombotic events, as described below.

At a histopathological level, arterial involvement by the thrombotic process is evident in the septal vessels of the lungs of the covd-19 patient, and also includes areas not affected by destructive inflammatory processes. Immunohistochemical staining of CD41 (endothelial marker) confirmed severe endothelial damage, water-soluble cytoplasm with cell shrinkage, denaturation, and lymphocyte adhesion on the endothelial surface, as shown in fig. 41A-D.

FIGS. 41A-D show representative images of immunohistochemical analysis of tissue sections taken from patients with COVID-19, showing vascular lesions in these patients.

FIG. 41A shows representative images of immunohistochemical analysis of septal vascular tissue sections from the lungs of a COVID-19 patient. As shown in fig. 41A, there is arterial involvement in the septal vessels of the lungs through the thrombotic process; note the initial organization of thrombus in the arterial lumen (H & E,400 x).

FIG. 41B shows representative images of immunohistochemical analysis of septal vascular tissue sections from the lungs of a COVID-19 patient. As shown in fig. 41B, similar pathological features as shown in fig. 41A are quite pronounced in most of the spaced blood vessels in the lung area that are not affected by the destructive inflammatory process (H & E,400 x).

FIG. 41C shows representative images of immunohistochemical analysis of tissue sections from medium diameter pulmonary septal vessels from a patient with COVID-19. As shown in fig. 41C, medium diameter pulmonary septal vessels (circled portions) with complete luminal thrombosis; immunohistochemical brown staining of CD34 (endothelial marker) confirmed severe endothelial damage, water-soluble cytoplasm with cell shrinkage, denaturation (see arrow on right side), and lymphocyte adhesion on endothelial surface (see arrow on left side).

FIG. 41D shows representative images of immunohistochemical analysis of liver parenchymal tissue sections from a patient with COVID-19. As shown in fig. 41D, vascular changes were also observed in the liver parenchyma, with large vessel part luminal thrombosis (H & E,400 x).

Identification and enumeration of Circulating Endothelial Cells (CECs)

Circulating Endothelial Cells (CECs) have been used as biomarkers of endothelial cell dysfunction (see Farinacci M et al, res Pract Thromb Haemost 3:49-58, 2019), and CEC counts have been shown to be elevated in sepsis-associated patients with ARDS as compared to sepsis-associated patients (Moussa M et al, intensive Care Med (2): 231-8, 2015). Results have also been published in the context of acute graft versus host disease (GvHD), where immune-mediated vascular endothelial cell attack causes it to detach from the vessel wall and mobilize into the blood stream (see, e.g., almii et al Bone Marrow Transplant 52:1637-1642, 2017).

Based on these preliminary observations and the findings in published acute graft versus host disease (GvHD), we began measuring CEC counts in a non-study cohort of randomly selected molecular-confirmed covd-19 patients in our hospital prior to initiation of the study with naso Li Shan antibody. In this non-study cohort of 33 covd-19 patients, we found a significant increase in CEC/mL of peripheral blood (median 110, range 38-877) compared to healthy controls (median 7, range 0-37) (p=0.0004), as shown in fig. 42A.

In this study, CEC/ml numbers were measured in COVID-19 patients before and after anti-treatment with naloxone Li Shan. As noted above, it was interesting to determine that the CEC/ml number of peripheral blood (median 110, range 38-877) increased significantly in the independent cohort of covd-19 patients when compared to healthy normal subjects (median 7, range 0-37) (see fig. 42A). An increase in the number of CECs/ml was also confirmed in 6 patients selected for anti-treatment by nano-cable Li Shan (median 334, range 0-9315). A rapid decrease in CEC/mL number was recorded after the first two doses after treatment with naloxone Li Shan (median 92CEC/mL, range 18-460) and confirmed after the fourth dose (median 73, range 0-593), as shown in fig. 42B. It was further confirmed that the number of CECs/mL was also reduced after the sixth nano-cable Li Shan antibody dose (median 59, range 15-276) (data not shown in fig. 42B).

FIG. 42A graphically illustrates CEC/ml counts in a normal healthy control (n-6) as compared to CEC/ml counts in a COVID-19 patient (n=33) that is not part of this study. As shown in fig. 42A, it was determined that the number of CECs/ml increased significantly in this independent covd-19 patient cohort when compared to healthy normal subjects.

Figure 42B graphically illustrates CEC/ml counts in 6 patients selected for this study before (baseline) and after resistance to treatment with nascent Li Shan, with boxes representing values from the first quartile to the third quartile, horizontal lines showing median values, and whiskers indicating minimum and maximum values. As shown in fig. 42B, increased CEC/ml was also confirmed in 6 patients selected for anti-treatment with naloxone Li Shan, which decreased rapidly after anti-treatment with naloxone Li Shan.

Because our hospital established guidelines for standard steroid use for covd-19 patients 16 days after the start of this study, steroid treatment was given to 5 of 6 patients as part of supportive therapy starting 2 to 10 days after the naloxone Li Shan anti-start. For this purpose, the number of CECs/ml was also assessed in a separate group of four patients receiving only steroids (all females, median age 83 years, ranging from 62 to 90 years, three requiring oxygen supply through the mask, and one at CPAP). In these four patients, CEC counts assessed after 48 hours were found to be unaffected by steroid administration (p=0.38). CEC counts were assessed at baseline and after 4 weeks of supportive care including steroid in two other steroid-only patients. In the first patient whose clinical course gradually worsens, the CEC count remains unaffected (271/mL versus 247/mL), while in the second patient, the clinical improvement is accompanied by a concomitant decrease in CEC (165 versus 65/mL).

CEC/mL increased significantly at baseline in 6 patients treated with nano-cable Li Shan (median 334, range 0-9315). CEC counts were rapidly decreased (p=0.01) using the naloxone Li Shan antibody following the second (median 92CEC/mL, range 18-460), fourth (median 72.5, range 0-593) and sixth (median 59, range 15-276) treatment doses. Serum concentrations of IL-6, IL-8, CRP and LDH were also significantly reduced using the halyard Li Shan anti-treatment, as described further below.

Serum levels of C-reactive protein (CRP), lactate Dehydrogenase (LDH) and cytokines

FIG. 43 graphically illustrates serum levels of C-reactive protein (CRP) (median; quartile range (IQR)) in 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after treatment with the naloxone Li Shan antibody. As shown in table 13, CRP serum levels in healthy subjects were in the range of (0.0-1.0 mg/dl) and CRP median levels in 6 covd-19 patients prior to initiation of treatment were 14mg/dl. As shown in fig. 43, CRP levels in 6 covd-19 patients decreased to near the median level of 0.0mg/dl, which was within the normal range of healthy subjects, after 2 weeks of anti-treatment with nano-cable Li Shan.

FIG. 44 graphically illustrates serum levels of Lactate Dehydrogenase (LDH) (median; IQR) in 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naline Li Shan. As shown in Table 13, the serum level of LDH in healthy subjects was in the range of (120-246U/l) and the median level of LDH in 6 COVID-19 patients prior to initiation of treatment was 518U/l. As shown in figure 44, LDH levels in covd-19 patients decreased to a median level of about 200U/l after 2 weeks of anti-treatment with nano-cable Li Shan, which was within the normal range of healthy subjects.

FIG. 45 graphically shows serum levels pg/mL (median; quartile range (IQR)) of interleukin 6 (IL-6) for 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naxol Li Shan. As shown in FIG. 45, at baseline prior to treatment, the median IL-6 level for the COVID-19 patient was about 180pg/mL. The median IL-6 level in the patient with COVID-19 was reduced to about 40pg/mL after 1 dose of naloxone Li Shan antibody (pre-dose 2), and further reduced to about 10pg/mL after 2 doses of naloxone Li Shan antibody (pre-dose 3).

FIG. 46 graphically illustrates serum levels pg/mL (median; quartile range (IQR)) of interleukin 8 (IL-8) for 6 COVID-19 patients at baseline prior to treatment (day 0), and at various time points after anti-treatment with Naxol Li Shan. Treatment with naloxone Li Shan antibody was given on days 1, 4, 7, 11 and 14. As shown in FIG. 46, at baseline prior to treatment, the median IL-8 level for the COVID-19 patient was about 30pg/mL. The median IL-8 level in the patient with COVID-19 decreased to about 20pg/mL after 1 dose of naloxone Li Shan antibody (pre-dose 2), and further decreased to about 15pg/mL after 2 doses of naloxone Li Shan antibody (pre-dose 3).

Clinical outcome after anti-treatment with naloxone Li Shan

The clinical characteristics of 6 patients selected for anti-treatment by nano-cable Li Shan are summarized in table 13. The median age was 56.5 years, and the majority of patients were men (83%). All patients were overweight or obese based on Body Mass Index (BMI) 25 or more and 30 or more. At the time of enrollment, all patients had pneumonia/ARDS where CPAP was required, with both patients rapidly worsening and requiring intubation shortly after enrollment. Treatment with naloxone Li Shan antibody was initiated within 48 hours from the onset of non-invasive ventilation with CPAP. A summary of the clinical results observed in these patients treated with the naloxone Li Shan antibody, updated to reflect the post-treatment patient status, is presented in table 14 below. The patient received twice weekly administration of naloxone Li Shan. After treatment, respiratory distress was improved in 4 patients (67%) and after the median of 3 nano-cable Li Shan anti-doses (range 2-3), they reduced ventilatory support from CPAP to high flow oxygen. Oxygen support was then reduced and stopped in 3 patients until discharge. Patient #4 developed massive pulmonary embolism on day 4 after initiation of treatment, as demonstrated by contrast-enhanced CT scan. For this, low molecular weight heparin was added on top of the underway nano-cable Li Shan antibody and rapid improvement of clinical and CT scan images was recorded after 7 days. In the last two patients (# 5 and # 6), rapid and progressive deterioration of severe ARDS was recorded shortly after enrollment. In case #5, severe ARDS (PiO thereof ₂ /FiO ₂ A value of 57) resulted in the patient being intubated on day 4. Nonetheless, subsequent clinical results are rapidly beneficial, and patients are discharged from the ICU after 3 days. After CPAP for 2 days, he is currently stable under low flow oxygen support. In case #6, severe ARDS developed after 4 days of enrollment and the patient needed intubation. She was put back in CPAP, similar to the previous case, and was then given a rapid clinical improvementPut into high flow oxygen and discharged later.

No treatment-related adverse events were reported in this study.

Table 14: patient outcome to date

* Reference is made to the update of patients 3-6 described below.

Discussion of the invention

Findings in this study indicate that endothelial injury and thrombosis are critical to the pathophysiology of covd-19-associated lung injury. Patients with severe respiratory failure demonstrated not only a significant increase in levels of CRP, LDH, IL-6 and IL-8, but also a significant increase in Circulating Endothelial Cells (CECs). This new observation is consistent with the histopathological findings detected in the lung and liver, which show significant endothelial damage and thrombosis in the covd-19 patient. Histopathological changes in multiple organ microvasculature, particularly microvascular thrombosis, similar to HSCT-TMA, further support the role of endothelial injury in COVID-19-related lung injury. Endothelial injury is known to be a key component of the pathophysiology of complement activation present in ARDS (Thompson BT et al, N Engl J Med,377 (6): 562-572, 2017). Complement activation has also been reported in models of SARS and MERS, and is important under other conditions characterized by endothelial injury. Vascular endothelial injury, increased capillary permeability in ARDS and root cause of pulmonary edema may also cause microvascular vascular disease and thrombosis.

The complement system is an important part of the immune system. Three pathways activate complement in response to different initiation events: classical pathway, lectin pathway and alternative pathway. The lectin pathway of complement is part of the innate immune response. Pattern recognition systems, the activation of the lectin pathway is initiated by members of the MASP enzyme family (MASP-1, MASP-2 and MASP-3). These proteases are synthesized as zymogens that form complexes in blood with lectins, in particular mannan-binding lectin (MBL), fiber-gelling proteins and collectins. These lectins recognize and bind to carbohydrate patterns found on the surface of pathogenic microorganisms or damaged host cells, targeting MASPs to their site of action and causing their activation. In this way, lectin pathway activation occurs on the surface of damaged endothelial cells. Lectin pathway activation was predicted to occur in the context of a covd-19-associated endothelial injury, as described in example 20.

MASP-2 is a key enzyme responsible for lectin pathway activation, and once activated, MASP-2 cleaves complement components 2 (C2) and C4, initiating a series of enzymatic steps leading to C3 and C5 activation, producing the formation of anaphylatoxins C3a and C5a, and C5b-9 (membrane attack complex). Preclinical, C3a and C5a have induced endothelial activation associated with endothelial injury and proinflammatory changes, leukocyte recruitment and endothelial apoptosis. Membrane-bound C5b-9 may also cause cell lysis. Even at sub-lysis, C5b-9 causes additional cellular damage which induces the secretion of pro-thrombotic factors, platelet activation, upregulation of adhesion molecules and morphological changes of dysfunction in the endothelium (Kerr H, richards A. Immunology 217 (2): 195-203, 2012). These complement-mediated activities can amplify endothelial injury and dysfunction, causing or exacerbating clinical conditions. Recent publications by Gao et al report a central involvement of the MASP-2 and lectin pathways in the pathophysiology of SARS and MERS in animal models. MASP-2, a key enzyme responsible for lectin pathway activation, binds to and undergoes activation by the COVID-19N protein (Gao et al, medRxiv 2020, 2020.03.29.20041962), and has been found in the microvasculature of lung tissue of severe COVID-19 patients (Magro C. Et al, transl Res 2020; doi.org/10.1016/j.trsl.2020.04.007). Activated MASP-2 initiates a series of enzymatic steps that lead to the production of anaphylatoxins C3a and C5a, and the formation of the membrane attack complex C5b-9 (Dobo et al, front Immunol 9:1851, 2018), which can induce a pro-inflammatory response and lead to cell lysis and death. MASP-2 may also cleave C3 directly through the C4 bypass (Yaseen S. Et al, FASEB J31 (5): 2210-9, 2017). Importantly, MASP-2 is located upstream of the lectin pathway, so inhibition of MASP-2 does not interfere with the cleavage set of the classical pathway (i.e.C1r/C1 s driven C3 and C5 convertases formation), preserving the adaptive immune response required against infection (Schwaebe et al Proc Natl Acad Sci (18): 7523-8, 2011).

In addition to its role in complement, MASP-2 acts directly on the coagulation cascade and contact system, cleaving prothrombin into thrombin and forming a fibrin clot (Gulla K.C., immunology 129 (4): 482-95, 2010; krarup A. Et al, PLoS One 2 (7): e623, 2007). As described in WO2019246367, incorporated herein by reference, the nano-cord Li Shan antibodies not only inhibit lectin pathway activation, but also block microvascular injury-related thrombosis, as well as MASP-2 mediated activation of kallikrein and factor XII. These activities may contribute to beneficial effects by inhibiting microvascular thrombosis, which may have played an important therapeutic role in patients with nano-cord Li Shan resistance therapy, particularly patients with massive pulmonary embolism. The nano-cord Li Shan anti-does not extend bleeding time, nor does it affect prothrombin or activated partial thromboplastin time, and bleeding is not observed in patients treated with the nano-cord Li Shan anti-treatment. While not wishing to be bound by any particular theory, it is believed that the nano-cord Li Shan resists coagulation (associated with factor XII activation) that may be due to endothelial damage, but does not block coagulation (factor VII driven) that is associated with extracellular matrix.

Lectin pathway inhibition has not been previously investigated as a treatment for covd-19. All patients in this study had covd-19 associated respiratory failure. In the current study, inhibition of the MASP-2 and lectin pathway by the naloxone Li Shan is associated with clinical improvement and survival of all COVID-19 patients treated with this drug. After anti-treatment with the MASP-2 inhibitor nano-cord Li Shan, all 6 patients recovered and were able to discharge. Clinical improvement was observed in patients with covd-19-associated respiratory failure following anti-treatment with naloxone Li Shan, which Li Shan anti-inhibits activation of MASP-2 and lectin pathways, further supporting the important role of the lectin pathway in the pathophysiology of covd-19. As described in this example, all 6 COVID-19 patients demonstrated clinical improvement after the Naso Li Shan anti-treatment. In each case, covd-19 lung injury had progressed to ARDS before nano-cable Li Shan anti-treatment, and all patients received non-invasive mechanical ventilation, each initiated at admission. After the first nano-cord Li Shan anti-dose, both patients experienced sustained deterioration and required invasive mechanical ventilation. Subsequently, the two patients were able to completely discontinue mechanical ventilation after treatment with continued nano-cable Li Shan resistance. Two patients (one cannulated and the other at CPAP) experienced massive bilateral pulmonary embolism and were fully resumpted with the nano-cable Li Shan resistance, which may benefit from the anticoagulant effect of the drug. The temporal pattern of laboratory markers (CEC, IL-6, IL-8, CRP and LDH) was consistent with the observed clinical improvement, and the proposed mechanism of action of the nano-cable Li Shan antibody. In particular, CEC counts appear to be a reliable tool for assessing endothelial damage and therapeutic response in this disease. Notably, improvements in IL-6 and IL-8 levels were also correlated in time with Naxomab treatment, suggesting that lectin pathway activation may precede cytokine storm elevation in COVID-19, and lectin pathway inhibition has potentially beneficial effects on cytokine storm in patients infected with COVID-19. (Xiong Y et al Emerg Microbes Infect 9 (1): 761-770, 2020). Two weeks of administration of naloxone Li Shan was initially planned, but when administration was first discontinued, the administration increased to 3 to 4 weeks after CEC elevation in patient # 1. After 3 to 4 weeks of nano-cable Li Shan anti-treatment, rebound pulmonary signs and symptoms were not observed. We did not see evidence of impaired viral defense in patients treated with the naloxone Li Shan antibody nor did we observe adverse events associated with the naloxone Li Shan antibody. Notably, the nano-cord Li Shan antibodies do not inhibit alternative or classical complement pathways and do not interfere with adaptive immune responses or antigen-antibody complexation. No evidence of the resistance of nano-cable Li Shan to the associated risk of infection was observed in the clinical trial. In addition to inhibiting lectin pathway activation, nano-cable Li Shan has also been shown to block MASP-2 mediated cleavage of prothrombin to thrombin (Krarup A et al, PLoS One;2 (7): e623, 2007), activation of kallikrein, and self-activation of factor XII to XIIa. These activities may contribute to beneficial effects by inhibiting microvascular thrombosis. The nano-cord Li Shan resists and does not extend bleeding time nor does it affect prothrombin or activate partial thromboplastin time. (Krarup PLoS One 2007)

The results described in this example strongly suggest that MASP-2 mediated lectin pathway activation by endothelial injury is implicated in the pathophysiology of COVID-19-related lung injury. Improvements in clinical status and laboratory findings after naloxone Li Shan anti-treatment are notable. These findings strongly suggest a meaningful clinical efficacy with supporting evidence concerning the mechanism of action of the drug and pathophysiology of the disease. Inhibition of the lectin pathway by the naloxone Li Shan antibody appears to be a promising potential treatment for covd-19 related lung injury.

Supplementary data from the clinical study described in this example

As described in this example, 6 laboratory-confirmed patients of COVID-19 and ARDS (according to Berlin standards) were treated twice weekly with naloxone Li Shan antibody (4 mg/kg Intravenous (IV)) for 3 to 4 weeks. All patients received standard supportive care including preventive enoxaparin (Clexane, sanofi Aventis) 4,000IU/0.4mL, azithromycin (Zitromax, pfizer SpA, italy) 500mg once a day, hydroxychloroquine (Plaquenil, sanofi Aventis) 200mg twice a day, and darunavir and cobalastat (Rezolsta, janssen-Cilag s.p.a., italy) 800/150mg once a day. Starting at day 3 and 27, all Covid-19 patients in the hospital received methylprednisolone (1 mg/kg) administered to 5 of the 6 patients treated with the naloxone Li Shan antibody according to the latest institutional guidelines.

Histopathological evaluation was performed on deceased covd-19 patients who were not treated with naloxone Li Shan. Clinical and laboratory measurements, including blood count, LDH and CRP levels, were collected for patients treated with the naloxone Li Shan and patients untreated with the naloxone Li Shan. Conventional blood tests were collected prior to each of the nano-cord Li Shan antibody doses and then twice weekly. Circulating endothelial cell counts and IL-6 and IL-8 levels were continuously assessed by flow cytometry. Respiratory function was assessed daily. All patients received chest Computed Tomography (CT) at admission to record interstitial pneumonia, if indicated clinically, pulmonary embolism during hospitalization. Chest radiography was also performed as indicated clinically.

The data is presented as a frequency with percentages of the classification variable, and as a median with a range for the continuous variable. Clinical and laboratory measured differences between time points were assessed using a nonparametric friedemann test. Pairing comparisons are performed using paired wilcoxon symbol rank test. Significance was fixed at 5%. Analysis was performed using R software (version 3.6.2).

Necropsy was performed on an initial group of 20 deceased covd-19 patients as described in this example. Consistent with the clinical manifestations of frequently fatal pulmonary thromboembolism, it was found that the lungs and liver of most patients are widely affected by embolism. Histologically, arterial embolism is evident in the septal vessels of the lungs, including areas not affected by destructive inflammatory processes. Immunohistochemical staining of CD34 (endothelial marker) confirmed severe endothelial damage with cell shrinkage, denatured water soluble cytoplasm and lymphocyte adhesion to endothelial surface as shown in figures 41A-D.

As described in this example, the lectin pathway that inhibits complement via nano-cable Li Shan correlates with clinical improvement in this study. Treatment with the nasucasian Li Shan antibody was associated with rapid and sustained CEC reduction, in parallel with concomitant reductions in serum IL-6, IL-8, CRP and LDH. In particular, CEC counts appear to be a reliable tool for assessing endothelial damage and therapeutic response in this disease. The temporary improvement of IL-6 and IL-8 treatment with Naline Li Shan suggests a potential beneficial effect on cytokine storm described in Covid-19 infected patients ((Xiong Y et al Emerg Microbes Infect (1): 761-770, 2020)) this finding indicates that endothelial injury is critical to the pathophysiology of COVID-19-related lung injury.

Two weeks of dosing were initially planned, but when dosing was discontinued for the first time, dosing increased to 3-4 weeks after CEC elevation in patient # 1. With the third week of administration, the CEC count of the patient was again improved. After 4 weeks of nano-cable Li Shan anti-treatment, rebound pulmonary signs and symptoms were not observed. We did not see evidence of impaired viral defense in patients treated with nano-cable Li Shan. Notably, the nano-cord Li Shan antibodies do not inhibit the classical or alternative complement pathways and do not interfere with the adaptive immune response or antigen-antibody complexation. No evidence of the resistance of nano-cable Li Shan to the associated risk of infection was observed in the clinical trial. In addition to inhibiting lectin pathway activation, nano-cord Li Shan has also been shown to block MASP-2 mediated cleavage of prothrombin to thrombin, activation of kallikrein, and self-activation of factor XII to XIIa. These activities may contribute to beneficial effects by inhibiting microvascular thrombosis, and this may have played an important therapeutic role, particularly in those patients with massive pulmonary embolism. The nano-cord Li Shan resists and does not prolong bleeding time, nor does it affect prothrombin or activated partial thromboplastin time, and bleeding is not observed in our treated patients.

Our findings strongly suggest that lectin pathway activation by endothelial injury is implicated in the pathophysiology of Covid-19-related lung injury. The lectin pathway by which complement was inhibited by nano-cable Li Shan correlated with clinical improvement in all patients in this study. The naloxone Li Shan resistance was well tolerated and no adverse drug reactions were reported. All patients improved and survived the course of treatment. Improvements in clinical status and laboratory findings after naloxone Li Shan anti-treatment are notable. These findings strongly suggest a meaningful clinical efficacy with supporting evidence concerning the mechanism of action of the drug and pathophysiology of the disease. Inhibition of the lectin pathway by the nano-cable Li Shan antibody appears to be a promising potential treatment for Covid-19 related lung injury.

Additional data is provided from the clinical study described in this example.

The clinical profile of 6 patients treated with naloxone Li Shan is summarized in table 13. The naloxone Li Shan anti-4 mg/kg was administered intravenously twice a week for 3 to 4 weeks. After treatment, all patients were clinically improved.

Of the 4 patients, enoxaparin was administered at therapeutic doses (100 IU/kg, twice daily) due to pulmonary embolism recorded by CT scan (patients #4 and # 6), medical decision (patient # 3) and rapid deterioration of respiratory function requiring intubation (patient # 5). Median follow-up was 27 days (16-90), and patients were given naloxone Li Shan anti twice weekly, with a median of a total of 8 naloxone Li Shan anti doses (range 5-8). All patients were clinically improved after treatment. Four patients (67%) reduced ventilatory support from CPAP to high flow oxygen (non-circulating ventilator or venturi oxygen mask) after a median of 3 nano-cable Li Shan anti-doses (range 2-3).

Figure 50 graphically illustrates the clinical results of 6 covd-19 infected patients treated with naloxone Li Shan.

As shown in figure 50, of 3 of these patients, oxygen support was withdrawn and then stopped, and they were discharged after the median of 6 (5-8) halyard Li Shan anti-total doses.

In patient #4, a large bilateral pulmonary embolism was recorded by contrast-enhanced CT scan 4 days after enrollment. Enoxaparin was added to the ongoing administration of naloxone Li Shan and after 11 days, rapid clinical and radiographic (repeated CT scan) improvement was recorded (fig. 47A and 47B) and subsequently discharged.

Among the remaining 2 patients (# 5 and # 6), rapid and progressive worsening of heavy ARDS was recorded shortly after enrollment.

In the patientIn #5, severe ARDS (PaO) ₂ /FiO ₂ 55) resulted in intubation on day 4. Nevertheless, the subsequent clinical outcome is rapidly favourable and the patient is discharged from the intensive care unit after 3 days. After CPAP for 2 days, he was stabilized with low flow oxygen support. Subsequently, he does not need oxygen and is discharged.

Patient #6 had PaO2/FiO2 60 and severe ARDS at enrollment and required intubation after 2 days. Her course is complicated by massive bilateral pulmonary embolism and hospital methicillin-resistant staphylococcus aureus (MRSA) infection. Her condition improved and after 18 days she pulled out, incised the trachea (due to claustrophobia) and was supported by low flow of oxygen. Her condition improved, oxygen support was removed, and on day 90 she was discharged. (days 33 to 90 are not shown in FIG. 50)

No treatment-related adverse events were reported in this study.

As described above, in patient #4, a large bilateral pulmonary embolism was recorded by contrast-enhanced CT scan 4 days after enrollment. Enoxaparin was added to the ongoing administration of naloxone Li Shan and after 11 days, rapid clinical and radiographic (repeated CT scan) improvement was recorded as shown in figures 47a, b.

Fig. 47A and 47B are images of CT scans obtained from the lungs of patient #4 with covd-19 pneumonia treated with naloxone Li Shan.

Figure 47A shows a CT scan of patient #4 at day 5 after enrollment (i.e., after anti-treatment with naloxone Li Shan), where the patient was observed to have severe interstitial pneumonia with diffuse ground glass shadows involving both the peripheral and central areas. Especially in the lower lobe of the left lung. Massive bilateral pulmonary embolism is accompanied by filling defects in the intercostal and segmental arteries (not shown).

Figure 47B shows a CT scan of patient #4 at day 16 after enrollment (i.e., after anti-treatment with nasosol Li Shan), with a significant decrease in ground glass shadows with almost complete regression of the substantial solid changes. In particular, "lithotripsy" signs with peripheral distribution were observed in the lower lobes. Obvious mediastinal emphysema. Minimal filling defects of the sub-segmental arteries of the right lung (not shown).

Figure 48 graphically illustrates serum levels of IL-6 (pg/mL) at baseline and at various time points after naloxone Li Shan anti-treatment (after 2 doses, after 4 doses) in patients treated with naloxone Li Shan anti-treatment. The boxes represent the values from the first quartile to the third quartile, the horizontal line showing the median value and the dots showing the values for all patients. FIG. 48 provides an update of the IL-6 data presented in FIG. 45.

Figure 49 graphically shows serum levels of IL-8 (pg/mL) at baseline and at various time points after naloxone Li Shan anti-treatment (after 2 doses, after 4 doses) in patients treated with naloxone Li Shan anti-treatment. The boxes represent the values from the first quartile to the third quartile, the horizontal line showing the median value and the dots showing the values for all patients. FIG. 49 provides an update of the IL-8 data presented in FIG. 46.

Figure 50 graphically illustrates the clinical results of 6 covd-19 infected patients treated with naloxone Li Shan. The bar color indicates different oxygen support (CPAP: yellow; mechanical ventilation with cannula: red; non-circulating respirator oxygen mask: green; low flow of oxygen through nasal cannula: light green; room air: blue). The naso Li Shan antibody dose is marked by blue arrows. The black circles indicate the onset of steroid therapy. Diamond symbols indicate TEP. Asterisks indicate discharge. CPAP = continuous positive airway pressure. NRM = non-circulating respirator oxygen mask. VM = venturi mask. TEP = pulmonary thromboembolism.

FIG. 51A graphically illustrates serum levels (units/liter, U/L) of aspartate Aminotransferase (AST) before and after nano-wire Li Shan anti-treatment. The black lines represent the median and quartile spacing (IQR). The red line represents normal levels, while the dots show the values for all patients.

Figure 51B graphically illustrates the serum levels of D-dimer values (ng/ml) in four patients whose baseline values were available prior to initiation of anti-treatment with nano-cable Li Shan. The black circles indicate when steroid therapy is initiated. The red line represents normal levels.

In summary, in this study, lectin pathway inhibitors were first used to treat covd-19, and 6 covd-19 patients with ARDS requiring Continuous Positive Airway Pressure (CPAP) or intubation received naloxone Li Shan resistance. The median age of the patients was 57 years (range 47-63 years), 83% were men, and all had comorbidities. At baseline, circulating Endothelial Cell (CEC) counts and serum levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP), lactate Dehydrogenase (LDH), D-dimer, and aspartate Aminotransferase (AST) -all markers of endothelial/cell damage and/or inflammation were significantly elevated. The nano-cable Li Shan anti-treatment was started within 48 hours after mechanical ventilation was initiated. The administration is twice a week for two to four weeks.

Results of the study

All patients resistant to treatment with nano-cable Li Shan were fully recovered, survived and discharged

Naline Li Shan anti-treatment is associated with rapid and sustained reduction/normalization of endothelial/cell damage and/or inflammation markers-CEC, IL-6, IL-8, CRP LDH, D-dimer and AST-across all assessments

The time pattern of the omicronlaboratory markers was consistent with the observed clinical improvement

In particular, CEC counts appear to be a reliable tool to assess endothelial damage and therapeutic response in this disease

The temporary improvement of the anti-treatment of IL-6 and IL-8 with nano-rope Li Shan suggests that lectin pathway activation may precede cytokine elevation in covd-19 and that lectin pathway inhibition has a beneficial effect on cytokine storms in patients infected with covd-19

The course of two patients (one cannulated and the other at CPAP) is further complicated by massive bilateral pulmonary embolism, and the two patients are resistant to full recovery using the nano-wire Li Shan, which may benefit from the anticoagulant effect of the drug

Naloxone Li Shan resistance was well tolerated in the study and no adverse drug reactions were reported

Two control groups with similar inclusion criteria and baseline characteristics, both showing a high mortality rate of 32% and 53%, were used for retrospective comparison.

Conclusion(s)

As demonstrated in this example, the anti-complement inhibiting lectin pathway with nano-cord Li Shan may represent an effective treatment for Covid-19 patients by reducing Covid-19 related endothelial cell damage and thus reducing the risk of inflammatory states and thrombosis. Lectin pathway inhibition has not been previously investigated as a treatment for covd-19. All patients in this study had a covd-19 related respiratory failure. After anti-treatment with the MASP-2 inhibitor, naloxone Li Shan, all patients recovered and were able to discharge, further supporting the importance of the lectin pathway in the pathophysiology of COVID-19.

The use of other complement inhibitors in covd-19 has been reported. ACOM-101, a C3 inhibitor based on compstatin (Mastaglio S. Et al, clin Immunol 215:108450, 2020) was used in one patient and elkuzumab was administered to four patients along with antiviral and anticoagulant therapy (Diuro F. Et al, eur Rev Med Pharmacol Sci (7): 4040-7, 2020) these five patients were CPAP and survived, two COVID-19 patients at high flow nasal oxygen received C5a antibodies combined with supportive therapy, including antiviral therapy, followed by steroid therapy, and these two patients survived, these reports together supported our discovery of the Naline Li Shan antibody however, unlike C3 and C5 inhibitors, MASP-2 antibody Naline Li Shan antibody fully maintained classical complement pathway function and did not interfere with adaptive immune responses or antigen-antibody complex mediated cleavage responses (Schwaeble W. Et al, proc Natl Acad Sci 108 (18): 7523-8, 2011) anti-clinical and no evidence of the infection of the Naline 5634 was observed in the clinical trial of the Naline.

While this was a single set of studies used in the same sense, two different control groups provided retrospective comparisons. The first is described in the paper recently published by Gritti et al (medRxiv 2020: 2020.04.01.20048561) which evaluates the use of the IL-6 inhibitor, setuximab in patients with COVID-19. The steuximab study and our nasoxazine Li Shan resistant study share the same major researchers (g.g. and a.r.), inclusion criteria and patient characteristics (i.e. demographics, symptoms, co-illness, ARDS severity, laboratory values and respiratory support at the time of inclusion). In this study, mortality in the steuximab treated group and the control group was 33% and 53%, respectively. The second retrospective comparator was represented by 33 patients, who were randomly selected in our hospital to evaluate the feasibility of CEC measurements in the covd-19 patients. Of these 33 patients, 22 met the same inclusion criteria as the patient treated with the naloxone Li Shan antibody and had similar baseline characteristics. However, the median baseline CEC counts were 101/mL versus 334/mL, respectively, for the control group compared to the nasucasian Li Shan anti-treated group. Interestingly, 20 of these 22 patients (91%) were treated with IL-6 inhibitors (tolizumab or stetuximab) and/or steroids, and this group had an overall 30-day mortality of 32%. When the outcome analysis was limited to 16 patients (median 58 years, ranging from 51-65 years) matched to the age of the patient treated with the naloxone Li Shan antibody, mortality was still 31%. In the latter group, 94% received IL-6 and/or steroid treatment and the median baseline CEC count at 55/mL was one-6 of the patients treated with naloxone Li Shan.

The use of steroids in COVID-19 has led to the reporting of mixed results (Veronese N. Et al, front Med (Lausanne) 7:170, 2020). More recently, randomized assessments of the covd-19 therapy (reciver) trial demonstrated that dexamethasone reduced 28-day mortality in patients under invasive mechanical ventilation by 28.7% (29.0% versus 40.7% with conventional care), in patients receiving oxygen support without non-invasive mechanical ventilation by 14% (21.5% versus 25.0% with conventional care), and had no effect on mortality in patients not receiving respiratory support at randomization (17.0% versus 13.2% with conventional care) (Horby p. Et al, medRxiv 2020:2020.06.22.20137273). Based on these data and our hospital experience, we believe that steroids play a role in treating patients with respiratory dysfunction, covd-19, in reducing inflammatory responses. In the group of naloxone Li Shan anti-treatment, 1 out of 6 patients (patient # 1) did not receive a steroid. Subsequently, in late March, institutional guidelines were updated, requiring all patients in our hospital to receive steroids. Of the 5 patients receiving the steroid resistant therapy to nano-cable Li Shan, 2 (patients #2 and # 3) initiated the therapy after having improved so that CPAP was no longer needed or discontinued the next day. As previously described, we assessed CEC counts in a separate group of four patients receiving steroid only for a short duration and found that the counts were not affected by steroid administration. This suggests that any beneficial effects of steroids on covd-19 related endothelial lesions may be delayed and have little effect on the recovery process in patients #2 and # 3.

Overall, our findings strongly suggest that endothelial injury-induced MASP-2 activation and lectin pathway play a key role in the pathophysiology of covd-19-related lung injury. Improvements in clinical status and laboratory findings after naloxone Li Shan anti-treatment are notable. These findings strongly suggest a meaningful clinical efficacy and provide supporting evidence concerning the mechanism of action of the drug and pathophysiology of the disease. Inhibition of the lectin pathway by the nano-wire Li Shan antibody appears to be a promising treatment for covd-19 related pulmonary and endothelial injury related embolism.

Further supplementary data from the clinical study described in this example

As described in this example, 6 laboratory-confirmed patients of COVID-19 and ARDS (according to Berlin standards) were treated twice weekly with naloxone Li Shan antibody (4 mg/kg Intravenous (IV)) for 3 to 4 weeks. As described in this example, all 6 patients in this study had COVID-19-related respiratory failure. After anti-treatment with the MASP-2 inhibitor nano-cord Li Shan, all patients recovered and were able to discharge. These patients have been monitored since discharge. By day 10 and 22 of 2020 (5 to 6 months after treatment with naloxone Li Shan) all 6 patients were clinically normal without evidence of any long-term sequelae that had been reported in the covd-19 patients not treated with naloxone Li Shan. Clinical laboratory measurements were also normal for all 6 patients by day 22 of 10 in 2020, including serum levels of D-dimer found to be within normal ranges (see table 15 below).

Table 15 shows baseline laboratory measurements obtained from 6 covd-19 infected patients at admission (baseline) prior to anti-treatment with naloxone Li Shan, compared to laboratory measurements obtained at 10 months in 2020 (after 5 to 6 months).

Table 15 summarizes the clinical characteristics of 6 nano-cable Li Shan anti-treated patients at baseline (prior to treatment, see also table 13) and as measured at 10 months in 2020 (5 to 6 months after treatment).

Table 15: laboratory measurements of COVID-19 patients treated with Nasog Li Shan

These results demonstrate that patients infected with naloxone Li Shan against treatment covd-19 in these 6 patients have resulted in complete recovery in these patients without evidence of any long-term sequelae.

As widely reported, many patients with covd-19 infection, including those with mild symptoms, as well as those with severe covd-19-related lung injury (e.g., ARDS and/or embolism), suffer from immediate complications from covd-19 infection, as well as long-term sequelae even after recovery from the initial infection, also known as "long-haul carriers". As described in Marshall M. ("The lasting misery of coronavirus long-hauls," Nature volume 585, pages 339-341, 9/17/2020), a person suffering from a more severe infection with COVID-19 may experience long-term damage in their lungs, heart, immune system, brain, central nervous system, kidneys, intestines and elsewhere, and even mild cases of infection with COVID-19 may cause persistent discomfort similar to chronic fatigue syndrome. Immediate and long-term sequelae from covd-19 infection include cardiovascular complications (including myocardial injury, cardiomyopathy, myocarditis, intravascular coagulation, stroke, venous and arterial complications, and pulmonary embolism), as further described in Marshall (2020); progression of neurological complications (including cognitive difficulties, confusion, memory loss also known as "brain fog", headache, stroke, dizziness, syncope, seizures, anorexia, insomnia, olfactory loss, gustatory loss, myoclonus, neuropathic pain, myalgia; neurological diseases such as Alzheimer's disease, geobatwo's syndrome, miller-Fisher syndrome, parkinson's disease); kidney injury (e.g., acute Kidney Injury (AKI)), pulmonary complications (including pulmonary fibrosis, dyspnea, pulmonary embolism); inflammatory conditions such as kawasaki disease, kawasaki-like disease, multisystem inflammatory syndrome in children; multiple system organ failure. See also Troyer A. Et al, brain, behavior and Immunity 87:43-39, 2020; babapore-Farrokhram S. Et al, life Sciences253:117723, 2020; and Heneka M. Et al, alzheimer's Research & Therapy, vol.12:69, 2020. As further described in yellow d et al Lancet Infect Dis 2020,9/1/2020, long-term complaints from persons recovering from acute covd-19 include: extreme fatigue, muscle weakness, low fever, inattention, memory errors, mood changes, sleep difficulties, needle pain in the arms and legs, diarrhea and vomiting, loss of taste and smell, sore throat and dysphagia, new episodes of diabetes and hypertension, rashes, shortness of breath, chest pain and palpitations.

As described in this example, patients infected with naloxone Li Shan against treatment of covd-19 in these 6 patients had resulted in complete recovery of these patients without evidence of any long-term sequelae from the covd-19 infection.

Example 22

OMS646 (Naxol Li Shan antibody) treatment in patient #7 infected with COVID-19

This example describes the use of naloxone Li Shan anti (OMS 646) in a seventh patient infected with covd-19 (patient # 7) using the methods described in example 20 and example 21. The results described in this example are consistent with those observed for patients infected with 6 covd-19 in example 21, and further confirm the efficacy of naloxone Li Shan against treatment of patients infected with covd-19.

Method and results：

Patient #7 was a 76 year old obese diabetic man infected with covd-19, with a long history of smoking and COPD, who had also undergone surgery for prostate cancer (i.e., classified as a "high risk" patient for covd-19 related complications). Patients enter the Bergamo hospital, initially requiring oxygen supply through nasal cannulae. His respiratory state rapidly worsens, first requiring oxygenation through the mask, followed by mechanical ventilation with continuous positive airway pressure, and then intubation. After intubation, treatment with a nano-cable Li Shan antibody (OMS 646) was initiated, the nano-cable Li Shan antibody (OMS 646) being a fully human monoclonal antibody consisting of immunoglobulin gamma 4 (IgG 4) heavy and lambda light chain constant regions. The nano-cord Li Shan antibodies bind with sub-nanomolar affinity and inhibit MASP-2. Patient #7 treated with naloxone Li Shan anti-treatment was administered intravenously twice a week at a dose of 4mg/kg for a total of 2 to 4 weeks, up to 6 to 8 doses (i.e., administration duration of two weeks, three weeks, or four weeks) according to the methods described in example 20 and example 21. To date, patient #7 has received 4 nano-cord Li Shan anti-doses. Patient #7 improved rapidly after anti-treatment with naloxone Li Shan, and he pulled out after the second dose. His laboratory findings are shown in figures 52A-E described below, with their dosing indicated by the vertical arrows on each figure.

Figure 52A graphically illustrates serum levels (ng/mL) of D-dimer values in critically ill patient #7 with covd-19 at baseline (day 0) prior to treatment, and at various time points after anti-treatment with naloxone Li Shan. Administration of the antibody with naloxone Li Shan is indicated by the vertical arrow. The red horizontal line represents the normal level.

Figure 52B graphically illustrates serum levels of C-reactive protein (CRP) in critically ill patient #7 with covd-19 at baseline (day 0) prior to treatment, and at various time points after anti-treatment with nano-wire Li Shan. Administration of the antibody with naloxone Li Shan is indicated by the vertical arrow. The red horizontal line represents the normal level.

Figure 52C graphically illustrates serum levels (units/liter, U/L) of aspartate Aminotransferase (AST) of critically ill patient #7 with covd-19 at baseline prior to treatment (day 0) and at various time points after naloxone Li Shan anti-treatment. Administration of the antibody with naloxone Li Shan is indicated by the vertical arrow. The red horizontal line represents the normal level.

Figure 52D graphically illustrates serum levels (units/liter, U/L) of alanine Aminotransferase (ALT) in critically ill patient #7 with covd-19 at baseline prior to treatment (day 0) and at various time points after naloxone Li Shan anti-treatment. Administration of the antibody with naloxone Li Shan is indicated by the vertical arrow. The red horizontal line represents the normal level.

Figure 52E graphically illustrates serum levels of Lactate Dehydrogenase (LDH) in patient #7 with severe covd-19 at baseline (day 0) prior to treatment, and at various time points after anti-treatment with nano-wire Li Shan. Administration of the antibody with naloxone Li Shan is indicated by the vertical arrow. The red horizontal line represents the normal level.

Summarizing results：

As shown in fig. 52A to 52E, patient #7 had high serum levels of D-dimer (considered to be the main marker of cureable property in covd-19), high serum levels of C-reactive protein (an inflammatory marker), high serum levels of aspartate aminotransferase (an enzyme marker of critical illness in covd-19), high serum levels of alanine aminotransferase (a liver function marker), and high serum levels of lactate dehydrogenase (a marker of cell death) at the time of admission and prior to anti-treatment with nano-cable Li Shan. As further shown in fig. 52A-52E, patient #7 was improved after the first naloxone Li Shan antibody dose, with all of the above laboratory measurements falling near or to normal levels after the fourth dose. He pulls out after the second halyard Li Shan anti-dose. After anti-treatment with naloxone Li Shan, the ICU staff was surprised for his rapid improvement. As described in example 21, the rapid improvement reported in this example for patient #7 was consistent with recovery of patient #1-6 infected with COVID-19 after anti-treatment with Naline Li Shan.

Additional data is provided from the clinical study described in this example:

as described in this example, patient #7 was improved after the first dose of nano-cable Li Shan, he was extubated after the second dose, and all of the above laboratory measurements were reduced to near normal or to normal levels after the fourth dose. As a renewal, patient #7 received a total of 6 nano-cable Li Shan anti-doses and was discharged. As shown in fig. 53, serological data from patient #7 over time indicated that appropriately high titers of anti-SARS-CoV-2 antibodies were generated during anti-treatment with nano-cable Li Shan, indicating that nano-cable Li Shan anti does not block the effector function of the adaptive immune response.

In addition to patients #1-7 described herein, numerous additional patients with codd-19 (total n=19) have been treated with naloxone Li Shan against 4mg/kg doses administered intravenously twice a week for 2 weeks to 4 weeks or 5 weeks, up to 4 to 10 doses (total two weeks, three weeks, four weeks or five weeks) under contemplative use according to the methods described in example 20 and example 21. All additional patients described in this example had severe disease of covd-19 related ARDS prior to treatment, all were intubated, most of which started naloxone Li Shan resistance several days after intubating, and all other therapies failed prior to starting naloxone Li Shan resistance. Surprisingly positive results were observed in most patients treated with the naloxone Li Shan antibody, similar to those observed for patients #1-7 described herein. Most of the covd-19 patients treated with the naloxone Li Shan anti-treatment showed rapid and significant improvement in clinical symptoms and laboratory values, and were subsequently discharged. Importantly, no clinical or laboratory evidence of long-term sequelae observed was shown for the patient of covd-19 who had a naloxone Li Shan anti-treatment available for their follow-up data (5-6 months after cessation of the naloxone Li Shan anti-treatment). It was also observed that surviving covd-19 patients treated with the halyard Li Shan antibody developed suitably high anti-SARS-CoV-2 antibodies as described above for patient # 7. These results demonstrate that treatment with the nano-cable Li Shan antibody (which specifically inhibits the lectin pathway and maintains the alternative and classical pathways of complement fully functional) preserves the anti-infective effector function of the adaptive immune response and maintains the antigen-antibody complex mediated lytic response, which plays an important role in killing virus infected cells.

A brief description of the course of treatment for critical illness COV-19-19 patients #8-15 treated with naloxone Li Shan in Bergamo, italy, and patients #1-4 treated with naloxone Li Shan in the united states is provided below:

patient #8 (Bergamo, italy)

Patient #8 was an obese man aged 76 with congestive heart failure, hypertension, dyslipidemia, and severe covd-19. He initiated naloxone Li Shan anti-treatment 3 days after intubation and died after the 3 rd dose from pre-existing cardiomyopathy complications. After 1 to 2 nano-cable Li Shan antibody doses, his D-dimer and LDH levels were improved. Serological data indicate that he did not develop high titers of anti-SARS-CoV-2 antibody.

Patient #9 (Bergamo, italy)

Patient #9 was a 41 year old overweight male with severe covd-19. He started the nano-cable Li Shan anti-treatment 2 days after intubation and pulled out after dose 2. He received a total of 6 doses and was discharged. After 1 to 2 nano-cable Li Shan antibody doses, his D-dimer and LDH levels were improved. Serological data indicated that he developed appropriately high titers of anti-SARS-CoV-2 antibody during the course of anti-treatment with Nasoh Li Shan.

Patient #10 (Bergamo, italy)

Patient #10 was an overweight male 65 years old with severe covd-19. He began a nano-cable Li Shan anti-treatment 3 days after intubation and pulled out after dose 4. He received a total of 9 nano-cable Li Shan antibody doses and was discharged. After 1 to 2 nano-cable Li Shan antibody doses, his D-dimer and LDH levels were improved. Serological data indicated that he developed appropriately high titers of anti-SARS-CoV-2 antibody during the course of anti-treatment with Nasoh Li Shan.

Patient #11 (Bergamo, italy)

Patient #11 was an overweight man 68 years old with hypertension, dyslipidemia and severe covd-19. He began the naloxone Li Shan anti-treatment 13 days after intubation. He received a total of 7 doses and died from multiple organ failure. Serological data indicate that he did not develop high titers of anti-SARS-CoV-2 antibody.

Patient #12 (Bergamo, italy)

Patient #12 was a 62 year old overweight man with diabetes, hypertension, dyslipidemia and severe covd-19. He began the naloxone Li Shan anti-treatment 2 days after intubation. He developed an nosocomial infection, requiring re-intubation followed by tracheotomy. He received a total of 6 doses of nano-cable Li Shan antibody and was discharged to the rehabilitation facility. Serological data indicate that he developed an appropriately high titre of anti-SARS-CoV-2 antibody during the course of anti-treatment with Nasoh Li Shan.

Patient #13 (Bergamo, italy)

Patient #13 was a 62 year old man with hypertension and severe covd-19. He began a nano-cable Li Shan anti-treatment 3 days after intubation and pulled out after 7 doses. He received a total of 8 doses of nano-cord Li Shan resistance and breathed spontaneously. Serological data indicate that he developed an appropriately high titre of anti-SARS-CoV-2 antibody during the course of anti-treatment with Nasoh Li Shan.

Patient #14 (Bergamo, italy)

Patient #14 was a 64 year old man infected with covd-19 with hypertension. He began a nano-cable Li Shan anti-treatment 6 days after intubation and pulled out after 7 doses. He received a total of 8 doses of nano-cable Li Shan resistance, initiated spontaneous breathing and discharged to the rehabilitation facility. Serological data indicate that he developed an appropriately high titer of anti-SARS-CoV-2 antibody during the course of anti-treatment with Nasoh Li Shan.

Patient #15 (Bergamo, italy)

Patient # was a 79 year old man with hypertension and severe covd-19. He began the naloxone Li Shan anti-treatment 3 days after intubation. He resists pulling out after 3 doses of nano-cable Li Shan and continues to improve. Serological data has not been obtained.

Patient #1 (USA)

Patient #1 was a 53 year old male with severe covd-19 who had been intubated for about 2 weeks after failure of other treatment regimens including adefovir, tolizumab, initial steroid treatment and convalescence plasma. He began treatment with naloxone Li Shan antibody and received both enoxaparin and methylprednisolone. He responded quickly and pulled out shortly after the 5 th nano-cable Li Shan anti-dose. He was sent to the rehabilitation facility for physical therapy, continued improvement and resumed work in the last month. He reported to have no long-term sequelae of covd-19.

Patient #2 (USA)

Patient #2 was a 55 year old african american woman with rapidly worsening respiratory function due to severe covd-19. She began treatment with naloxone Li Shan antibody a few days after intubation. Her oxygen demand has been successfully removed, but because of intolerance of the mask, tracheostomies are placed for low levels of oxygen support, and feeding tubes are inserted. She was sent to an emergency care facility and then returned home. Tracheostomies and feeding tubes were removed and she reported complete recovery without evidence of long-term clinical sequelae.

Patient #3 (USA)

Patient #3 was an 80 year old man with severe covd-19. He began treatment with naloxone Li Shan antibody a few days after intubation. He died after the 3 rd or 4 th nano-cable Li Shan anti-dose. His death was reported to be associated with complications associated with barotrauma and secondary to mechanical ventilation. His family refuses to receive in vitro membrane oxygenation (ECMO) therapy for religious reasons.

Patient #4 (USA)

Patient #4 was a 61 year old man with hypertension and severe covd-19. He had been intubated for 8 days and experienced ECMO before the initiation of the nano-cable Li Shan anti-treatment. He failed treatment with rad Weiba retenib and high doses of steroids. He has so far received several nano-cable Li Shan anti-doses and his clinical status remained stable.

Influenza virus

As described in examples 20, 21 and 22, the lectin pathway has been demonstrated to contribute to lung injury in COVID-19 infection, and the representative MASP-2 inhibitory antibody, nano-cable Li Shan, is effective in alleviating pulmonary symptoms in patients with COVID-19 infection. Complement activation has also been demonstrated to contribute to lung injury in influenza H5N1 virus infection models. Lung histopathological changes are very similar in patients with H5N1 infection and SARS-CoV infection. In the H5N1 murine model, expression of MASP-2RNA, C3a receptor RNA and C5a receptor RNA all increased on the first day after infection. Complement inhibition using C3aR antagonists or cobra venom factors reduces lung injury and clinical signs. Survival is also increased (see Sun et al Am J Respir Cell Mol Biol 49 (2): 221-30, 2013). Thus, MASP-2 inhibitors would also be expected to be useful in methods of treating, inhibiting, alleviating or preventing acute respiratory distress syndrome or other manifestations of a disease in a mammalian subject infected with influenza virus.

In accordance with the foregoing, in one aspect, the present invention provides a method for treating, inhibiting, reducing or preventing acute respiratory distress syndrome or other manifestations of a disease, such as thrombosis, in a mammalian subject infected with a coronavirus or influenza virus, comprising administering to the subject an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation (i.e., inhibit lectin pathway activation). In some embodiments, the subject has one or more respiratory symptoms and/or thrombosis, and the method comprises administering to the subject an amount of a MASP-2 inhibitor effective to ameliorate at least one respiratory symptom (i.e., improve respiratory function) and/or reduce thrombosis.

In one embodiment, the method comprises administering the composition to a subject infected with covd-19. In one embodiment, the method comprises administering the composition to a subject infected with SARS-CoV. In one embodiment, the method comprises administering the composition to a subject infected with MERS-CoV. In one embodiment, the subject is identified as having coronavirus (i.e., covd-19, SARS-CoV, or MERS-CoV) prior to administration of the MASP-2 inhibitor. In one embodiment, the subject is identified as infected with covd-19 and in need of oxygen supplementation, and a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, such as nano-cord Li Shan, is administered to the patient in a dose and for a period of time effective to eliminate the need for oxygen supplementation.

In one embodiment, the subject is identified as having covd-19 and has or is at risk of developing covd-19 induced thrombosis, and the method comprises administering a composition comprising a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, such as a nano-cord Li Shan antibody) in a therapeutically effective amount to treat, prevent, or reduce the severity of coagulation or thrombosis in the subject. In some embodiments, the methods of the invention provide anticoagulation and/or antithrombotic without affecting hemostasis. In one embodiment, the level of D-dimer is measured in a subject having a covd-19 to determine the presence or absence of thrombosis in the subject, wherein a level of D-dimer above a standard range is indicative of the presence of thrombosis, and the subject is treated with a therapeutically effective amount of a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody, such as a nano-cable Li Shan antibody) to treat, prevent, or reduce the severity of coagulation or thrombosis in the subject, which may be measured, for example, by a decrease in the level of D-dimer to a level within the normal range of a healthy subject.

In one embodiment, the method comprises administering the composition to a subject infected with an influenza virus, such as influenza a virus (H1N 1 (causing "spanish influenza" in 1918 and "swine influenza" in 2009 "), H2N2 (causing" asian influenza "in 1957), H3N2 (causing" hong kong influenza "in 1968), H5N1 (causing" avian influenza "in 2004), H7N7, H1N2, H9N2, H7N3, H10N7, H7N9, and H6N 1); or influenza b virus or influenza c virus. In one embodiment, the subject is identified as having influenza virus prior to administration of the MASP-2 inhibitor.

In one embodiment, the subject is determined to have an increased level of circulating endothelial cells in a blood sample obtained from the subject prior to treatment with the MASP-2 inhibitor as compared to the level of circulating endothelial cells of a control healthy subject or population. In some embodiments, the method comprises administering an amount of a MASP-2 inhibitor sufficient to reduce the number of circulating endothelial cells in a subject infected with a coronavirus or an influenza virus.

In one embodiment, the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% of human serum, IC thereof ₅₀ 30nM or less.

In some embodiments, the method comprises administering to a subject infected with a coronavirus or influenza virus a composition comprising a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:69, at a dose of 1mg/kg to 10mg/kg (i.e., 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, or 10 mg/kg), at least once per week (e.g., at least twice per week or at least three times per week), for a period of at least 2 weeks (e.g., at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 9 weeks, or at least 10 weeks, or at least 11 weeks, or at least 12 weeks).

In one embodiment, the dose of MASP-2 inhibitory antibody (e.g., naloxone Li Shan antibody) is administered to a subject with COVID-19 at a dose of about 4mg/kg (i.e., 3.6mg/kg to 4.4 mg/kg) for a period of at least two weeks, or at least three weeks, or at least four weeks (e.g., two to four weeks) at least twice a week.

In one embodiment, the method comprises administering intravenously a fixed dose of about 370mg (+ -10%) of MASP-2 inhibitory antibody to a subject infected with coronavirus or influenza virus twice weekly for a treatment period of at least 8 weeks.

In one embodiment, the subject has a covd-19 induced pneumonia or ARDS and the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered for a time sufficient to alleviate one or more symptoms of the pneumonia or ARDS. In one embodiment, the subject is on a mechanical ventilator and the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered at a dose and for a period of time sufficient to discontinue the need for mechanical ventilation. In one embodiment, the subject is on an invasive mechanical ventilator. In one embodiment, the subject is on a non-invasive mechanical ventilator. In one embodiment, the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered at a dose and for a period of time sufficient to discontinue use of supplemental oxygen.

In one embodiment, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered as monotherapy to a subject infected with coronavirus or influenza virus. In some embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered to a subject infected with coronavirus or influenza virus in combination with one or more additional therapeutic agents, e.g., in a composition comprising a MASP-2 inhibitor and one or more antiviral agents, or one or more anticoagulants, or one or more therapeutic antibodies, or one or more therapeutic small molecule compounds. In some embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered to a subject infected with a coronavirus or an influenza virus, wherein the subject is undergoing treatment with one or more additional therapeutic agents, such as one or more antiviral agents, or one or more anticoagulants, or one or more therapeutic antibodies, or one or more therapeutic small molecule compounds.

In accordance with the foregoing, in another aspect, the invention provides a method for treating, ameliorating, preventing or reducing the risk of a mammalian subject infected with coronavirus or influenza virus developing one or more long-term sequelae, the method comprising administering to the subject an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation (i.e., inhibit lectin pathway activation). In some embodiments, the subject has one or more respiratory symptoms and/or thrombosis, and the method comprises administering to the subject an amount of a MASP-2 inhibitor effective to ameliorate at least one respiratory symptom (i.e., improve respiratory function) and/or reduce thrombosis.

In one embodiment, a subject is identified as infected with covd-19 and experiencing mild symptoms, and a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, e.g., naloxone Li Shan antibody, is administered to the subject in a dose and for a period of time effective to treat, ameliorate, prevent or reduce the risk of developing one or more of the long-term sequelae associated with covd-19 in the subject. In some embodiments, the methods are useful for treating, ameliorating, preventing, or reducing the risk of one or more of the long-term sequelae associated with covd-19 or with one or more of the long-term sequelae associated with covd-19 in a subject having been infected with covd-19, wherein the long-term sequelae are selected from cardiovascular complications (including myocardial injury, cardiomyopathy, myocarditis, intravascular coagulation, stroke, venous and arterial complications, and pulmonary embolism) in a subject having been infected with covd-19; progression of neurological complications (including cognitive difficulties, confusion, memory loss also known as "brain fog", headache, stroke, dizziness, syncope, seizures, anorexia, insomnia, olfactory loss, gustatory loss, myoclonus, neuropathic pain, myalgia; neurological diseases such as Alzheimer's disease, geobatwo's syndrome, miller-Fisher syndrome, parkinson's disease); kidney injury (e.g., acute Kidney Injury (AKI)), pulmonary complications (including pulmonary fibrosis, dyspnea, pulmonary embolism), and inflammatory conditions such as kawasaki disease, kawasaki-like disease, childhood multisystem inflammation syndrome (MIS-C), and multisystem organ failure. Recently published data shows that SARS-CoV-2 infection in children results in a high incidence of TMA independent of clinical severity (see Diorio C. Et al, blood Advances volume 4 (23), dec 8, 2020). It has also been reported that SARS-CoV-2 infection in children can lead to multisystem inflammatory syndrome (MIS-C) (see Radia T. Et al, paediatr Respri Rev, 8/11/2020).

More than 60% of "restored" covd-19 patients have recently been reported by various international communities to have severe sequelae, including cognitive/CNS, lung, heart, liver and other abnormalities (see Bonow et al, jamacarioglobin volume 5 (7) 7, month 7, del Rio et al, volume 324 (17), month 11, 2020, lindner et al, jamacarioglobin volume 5 (11), month 11, 2020, marchiano s et al, bioRxiv, month 8, 30, puntmann v et al, jamacariology volume 5 (11), month 11, xong q. Et al, clin Microbial Infect). For example, as described in yellow D et al, lancet Infect Dis 2020,9/1/2020, long-term complaints from persons recovering from acute COVID-19 include: extreme fatigue, muscle weakness, low fever, inattention, memory errors, mood changes, sleep difficulties, needle pain in the arms and legs, diarrhea and vomiting, loss of taste and smell, sore throat and dysphagia, new episodes of diabetes and hypertension, rashes, shortness of breath, chest pain and palpitations. Notably, as described in examples 21 and 22 herein, no clinical or laboratory evidence of long-term covd-19 sequelae was shown with respect to 5 to 6 months follow-up of covd-19 patients treated with naloxone Li Shan in the first 6 Bergamo studies.

In one embodiment, the MASP-2 inhibitor is an inhibitor of MASP-2 expression.

In one embodiment, a dose of MASP-2 inhibitory antibody (e.g., naloxone Li Shan antibody) is administered to a subject with COVID-19 at a dose of about 4mg/kg (i.e., 3.6mg/kg to 4.4 mg/kg) for a period of at least two weeks, or at least three weeks, or at least four weeks, or at least five weeks, or at least six weeks, or at least seven weeks, or at least eight weeks (e.g., two weeks to four weeks, or two weeks to five weeks, or two weeks to six weeks, or two weeks to seven weeks, or two weeks to eight weeks) at least twice a week.

In one embodiment, the subject has a covd-19 induced pneumonia or ARDS, and the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered for a time sufficient to reduce one or more symptoms of pneumonia or ARDS, and to reduce or prevent a long-term sequelae associated with covd-19. In one embodiment, the subject is on a mechanical ventilator and the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered at a dose and for a period of time sufficient to discontinue the need for mechanical ventilation. In one embodiment, the subject is on an invasive mechanical ventilator. In one embodiment, the subject is on a non-invasive mechanical ventilator. In one embodiment, the MASP-2 inhibitor (e.g., MASP-2 inhibitory antibody) is administered at a dose and for a period of time sufficient to discontinue use of supplemental oxygen.

In one embodiment, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered as a monotherapy to a subject infected with a coronavirus or an influenza virus. In some embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered to a subject infected with coronavirus or influenza virus in combination with one or more additional therapeutic agents, e.g., in a composition comprising a MASP-2 inhibitor and one or more antiviral agents, or one or more anticoagulants, or one or more therapeutic antibodies, or one or more therapeutic small molecule compounds. In some embodiments, a MASP-2 inhibitor (e.g., a MASP-2 inhibitory antibody) is administered to a subject infected with a coronavirus or an influenza virus, wherein the subject is undergoing treatment with one or more additional therapeutic agents, such as one or more antiviral agents, or one or more anticoagulants, or one or more therapeutic antibodies, or one or more therapeutic small molecule compounds.

Example 23

SARS-Cov-2 nucleocapsid (N) protein binds MASP-2 and activates complement C4, and representative MASP-2 inhibitory antibody HG4 inhibits this activation.

Background/principle:

as described in examples 21 and 22, anti-treatment of patients with the ARDS-covd-19 with the MASP-2 inhibitory antibody nano-Li Shan resulted in rapid improvement. This example shows that the SARS-Cov-2 nucleocapsid (N) protein binds MASP-2 and activates complement C4, and that a representative MASP-2 inhibitory antibody HG4 inhibits this activation, further confirming that the MASP-2-mediated lectin pathway is activated following SARS-Cov-2 infection.

SARS-Cov-2 nucleocapsid protein binding MASP-2

The method comprises the following steps:

the microtiter plates were coated with 1. Mu.g/well recombinant SARS-Cov-2 nucleocapsid protein (NP 2) or control matrix (BSA). The residual binding sites were blocked with 1% bsa. Serial dilutions of recombinant MASP-2 (rmsp-2) were added and binding was detected using anti-MASP-2 mAb.

Results:

FIG. 54 graphically illustrates the concentration-dependent binding of recombinant MASP-2 to SARS-Cov-2 nucleocapsid protein (NP 2) as compared to a BSA control.

MASP-2 binds directly to SARS-Cov-2N protein and mediates complement C4 activation

The method comprises the following steps:

the microtiter plates were coated with 2.5. Mu.g/well SARS-Cov-2 recombinant nucleocapsid protein (NP 2). The residual binding sites were blocked with 1% BSA. rMASP-2 (1 μg) in Barbital Buffered Saline (BBS) was added. Control wells received buffer only. After incubation for 1 hour at 37℃the wells were washed with TBS/Tween. Purified human C4 (1 μg) was added to each well. MASP-2 inhibitory antibody HG4 (0.1. Mu.M) (also known as OMS646-SGMI-2, as described in example 13) was added to certain wells coated with NP2 containing rMASP-2 and C4. After 1 hour incubation at 37 ℃, the supernatant was aspirated and separated on SDS-PAGE under reducing conditions and loaded on a Western blot as follows:

Lane 1: c4 control only

Lane 2: NP2 plus rMASP-2 plus C4:

lane 3: NP2 plus rMASP-2 plus C4 plus HG4 (0.1 mM)

Lane 4: NP2 plus C4

Lane 5: BSA plus rMASP-2 plus C4

Results:

FIG. 55 depicts an SDS-PAGE Western blot gel, wherein: lane 1 contains purified C4 as a control, showing bands corresponding to c4α, c4β and c4γ. Lane 2 contains NP2 plus rMASP-2 plus C4, showing C4 alpha, C4 beta, C4 gamma and a new band corresponding to C4' alpha, indicating that MASP-2 binds directly to NP2 and cleaves C4. Lane 3 contains NP2 plus rMASP-2 plus C4 plus HG4 (0.1. Mu.M), indicating that the addition of MASP-2 inhibitory antibody HG4 inhibited NP2/MASP-2 mediated C4 cleavage. Lane 4 contains NP2 plus C4, indicating that in the absence of MASP-2, there is no C4 cleavage in the sequence. Lane 5 contains BSA plus MASP-2 plus C4, showing no C4 cleavage in the absence of NP 2.

Discussion of the invention

This example shows that SARS-Cov-2 nucleocapsid protein (NP 2) binds MASP-2 and activates complement C4, and that a representative MASP-2 inhibitory antibody HG4 inhibits this activation, further confirming that the MASP-2-mediated lectin pathway is activated following SARS-Cov-2 infection.

Example 24

Longitudinal study to measure complement activation in acute covd-19 patients compared to healthy volunteers

Background/principle:

infection with the new strain of coronavirus, SARS-CoV-2, is generally asymptomatic or has a slight exacerbation. However, in a few infected individuals, SARS-CoV-2 can cause severe to life-threatening diseases with mild to severe long-term morbidity and mortality. The factors that determine the susceptibility to severe acute exacerbations are not entirely clear, and in addition to complications, genetic factors, epigenetic phenomena, and age and sex differences are thought to influence the risk of developing severe to fatal pathology. Experimental evidence provided herein and reported in Rambaldi A. Et al, immunobiology 225 (6): 152001,2020 and elsewhere clearly indicate that the complement system is a key driver of inflammatory response in the initiation and maintenance of endothelial pathology of acute COVID-19. Treatment of critically ill covd-19 patients with a naloxone Li Shan anti (MASP-2 inhibitory antibody) as described in examples 21 and 22 herein and as reported in Rambaldi a. Et al, immunobiology 225 (6): 152001,2020, achieved therapeutic breakthrough and disease manifestations improved rapidly after infusion. Consistent with the therapeutic efficacy of the nano-cable Li Shan antibody, it was demonstrated that SARS-Cov-2 nucleocapsid protein (NP 2) directly binds MASP-2, resulting in C4 cleavage, which is blocked by MASP-2 antibody HG4, as further described in example 23.

In this example, further studies of each of the three complement activation pathways were examined in donors at defined stages and severity of covd-19 in order to identify clinical and prognostic markers for acute covd-19, to identify windows of therapeutic opportunity for treatment, and to gain further insight into molecular events that lead to acute covd-19. The results may also provide predictive clinical markers for disease severity and sustained pro-inflammatory events (which lead to adverse consequences of long-covd-19 syndrome).

The method comprises the following steps:

this example describes preliminary results from an ongoing study measuring complement activation in subjects of different classes, where longitudinal plasma and serum samples were collected from donors of different classes, as follows:

category:

(1) Donors with acute COVID-19 or post-acute COVID-19 (also known as long-term COVID-19),

acute patient: samples of COVID-19 patients were collected within 15 days (0-4 days; 5-10 days and 11-15 days) after admission.

Recovery/convalescence patient: subjects 3 months after recovery from acute covd-19 (i.e., patients who survived and were discharged in acute covd).

(2) SARS-CoV-2 test is positive and asymptomatic or slightly asymptomatic donors without hospitalization.

(3) Uninfected Health Care Workers (HCW) (i.e., SARS-CoV-2 seronegative)

Complement determination (CH 50, C5a, bb)

Complement activation results in release of pro-inflammatory anaphylatoxin C5a as well as factor Bb (alternative pathway activation marker), which is measured in different subject populations, as described below.

CH50 determination

CH50 assay measures total complement hemolysis of sheep erythrocytes coated with anti-sheep erythrocyte antibodies.

Figure 56 graphically illustrates CH50 values in samples obtained from different subject populations in a longitudinal study, wherein each "x" symbol in the figure represents an individual subject.

As shown in fig. 56, total complement hemolysis was compromised by early complement depletion at SARS-CoV-2 infection, as demonstrated below: the CH50 values for acute COVID-19 patients were very low, 0-4 days after admission, 5-10 days after admission and 11-15 days after admission, compared to the CH50 values for convalescent patients (2-3 months after admission), SARS-Cov-2 positive and seronegative (i.e., SARS-Cov-2 negative). As further shown in fig. 56, most convalescent patients showed an increase in CH50 values back into the normal range.

C5a assay

C5a assay measures the proinflammatory complement activation product C5a that is common between all three complement pathways. The assay was a commercially available sandwich ELISA (catalog # DY 2037) from R & D Systems.

Figure 57 graphically illustrates C5a levels (ng/ml) in plasma samples obtained from different subject populations in a longitudinal study, wherein each "x" symbol in the figure represents an individual subject.

As shown in fig. 57, the C5a levels in plasma obtained from acute covd-19 patients 0-4 days after admission (n=16), 5-10 days after admission (n=12) and 11-15 days after admission (n=12) were significantly higher than the C5a levels in plasma obtained from convalescent patients (n=36), seropositive persons (n=30) and seronegative persons (n=26).

Bb assay

The activation state of the Alternative Pathway (AP) was detected using a commercially available sandwich ELISA, which detects the neoepitope of the Bb activation product of AP (Quidel MicroVue Bb Plus EIA).

Figure 58 graphically illustrates the Bb levels (ug/mL) in plasma obtained from different subject populations in a longitudinal study, wherein each "x" symbol in the figure represents an individual subject.

As shown in fig. 58, the levels of Bb in plasma obtained from acute covd-19 patients at 0-4 days post-admission, 5-10 days post-admission, and 11-15 days post-admission were significantly higher than those obtained from convalescent patients, seropositive and seronegative persons. As further shown in fig. 58, the recovered patient's Bb levels were within the range of normal healthy controls (seronegative personnel).

Results:

as shown in figures 56, 57 and 58, complement activation occurred early in subjects with acute covd-19 as evidenced by low CH50 (figure 56), high C5a levels (figure 57), and high Bb levels (figure 58) in 15 days of admission for acute patients as compared to convalescent patients and healthy controls. It was further demonstrated that AP was activated early in infection, as demonstrated by the high Bb levels of acute patients within 15 days of admission, and reverted to normal levels after recovery (see figure 58).

Example 25

High levels of the C1-INH/MASP-2 complex are associated with acute COVID-19

Background/principle:

SARS-Cov2 is a emerging virus that has a very high risk of infectivity and mortality in those suffering from severe endothelial disease and respiratory symptoms. In order to maximize the success of protecting people from such diseases, biomarkers and highly accurate tests are urgently needed to identify those at risk of developing acute and/or long-term disease (post-acute covd-19, alternatively referred to as long-term covd-19 syndrome) or who have developed a protective immune response against the covd-19 disease response. Tests are also needed to determine the efficacy of therapeutic agents to treat and/or prevent covd-19 related complications, including those with or at risk of developing long-covd-19.

As described in example 24, complement activation occurred early in subjects with acute covd-19, as evidenced by low CH50 (fig. 56), high C5a levels (fig. 57), and high Bb levels (fig. 58) in acute patients within 14 days of admission as compared to healthy controls. It was further demonstrated that the Alternative Pathway (AP) was activated early in the infection, as demonstrated by the high Bb levels of acute COVID-19 patients within 15 days of admission, and reverted to normal levels after recovery (see FIG. 58).

This example describes the development of a sensitive sandwich ELISA assay capable of detecting the amount of MASP-2/C1-INH complex in a human serum sample. This example further describes the use of such a sensitive sandwich ELISA assay to interrogate the activation state of the Lectin Pathway (LP) in each group of subjects (i.e., acute COVID-19, convalescent patients and healthy control subjects) by measuring the level of liquid phase MASP-2/CI-INH complex in serum samples obtained from these subjects.

The method comprises the following steps:

MASP-2/C1-INH Complex ELISA assay

MASP-2 is found in plasma as a zymogen and is associated with one of several Lectin Pathway (LP) pattern recognition molecules. The zymogen form is loosely bound to the serine protease inhibitor Cl-INH. When sufficient LP recognition molecules are tightly bound to the activating surface, the zymogen MASP-2 is cleaved by another MASP-2 molecule or MASP-1 into two disulfide-linked chains. The cleaved MASP-2 is an active form of the enzyme that cleaves its substrate, downstream complement components C4 and C2. The activity of MASP-2 is regulated by C1-INH, which binds tightly to activated MASP-2, forming a stable 1:1 complex.

In order to determine the activation state of the LP effector enzyme MASP-2, use is made of the feature that exploits the fact that: once MASP-2 is activated, the C1 inhibitor (C1-INH) which acts as a pseudo-substrate forms a covalent liquid phase MASP-2/C1-INH complex. Thus, the level of MASP-2/C1-INH complex in plasma or serum samples provides a clear measure of recent LP activation.

The human MASP-2 protein (mature form) is shown in SEQ ID NO. 6.

Human C1 esterase inhibitor (C1-INH), genbank CAA38358, shown below as SEQ ID NO 86 (aa 1-21 signal peptide, mature protein aa 22-500)

Kajdacsi et al Front Immunol vol 11,2020 used anti-human MASP-2 monoclonal rat IgG1 (mAh 8B 5) from Hycult Biotech as a capture antibody to measure MASP-2/C1-INH complex in 10% serum concentration in healthy and Hereditary Angioedema (HAE) patients. Hansen et al, J of Immunol 195:3596-3604,2015 also used anti-human MASP-2 monoclonal rat IgG1 (mAb 8B 5) from Hycult Biotech in ELISA assays to measure MASP-2/C1-INH complexes in HAE patients. Hansen et al observed that MASP-2/C1-INH complex was detected only at very high human serum concentrations (20% or higher) compared to MASP-1/C1-INH complex, and considered this probably due to the fact that the serum concentration of MASP-2 was much lower compared to MASP-1, or due to the fact that: compared to the MASP-1mAb used in their study, the commercial MASP-2 mAb8B5 is less suitable as a detection antibody (see Hansen et al, pages 3602-3603, bridging paragraphs).

To develop a sensitive ELISA assay suitable for screening individual patient samples with serum concentrations below 10% (i.e., 0.3% to 8% serum, e.g., 0.3% to 7% serum, e.g., 0.3% to 6% serum, e.g., 0.3% to 5% serum) for the presence and/or amount of MASP-2/C1-INH complex, a panel of monoclonal antibodies (clones C1, C7, D8 and H1) known to bind MASP-2 are tested as capture antibodies. These mAbs (clones C1, C7, D8 and H1) were generated from hybridomas obtained from immunized MASP-2KO mice and were found to bind MASP-2 but were unable to inhibit MASP-2 functional activity (data not shown).

anti-MASP-2 mAb: c1, C7, D8, H1 were tested as candidate capture antibodies in ELISA assay formats for detection of MASP-2/C1-INH complexes as follows.

(i) Nunc Maxisorb microtiter plates with carbonate buffer (15 mM Na ₂ CO ₃ 、35mM NaHCO ₃ 100. Mu.l of anti-MASP-2 candidate capture antibody clones #C1, #C7, #D8 and #H2 1 (2. Mu.g/ml) in pH 9.6)) Coating was carried out overnight at 4 ℃. The microtiter plates were blocked with 280. Mu.l/well of 1% (w/v) BSA in TBS buffer for 1 hour at room temperature.

(ii) By combining Normal Human Serum (NHS) in a medium containing 5mM Ca ²⁺ Activated control serum was prepared by dilution to 20% v/v in Tris buffered saline (TBS; 10mM Tris-Cl,140mM NaCl,pH 7.4). mannan-Sepharose (100 uL, sigma catalog number M9917) was treated with five volumes of TBS/Ca ²⁺ Washed twice and resuspended to 100 μl in the same buffer. Mu.l of 20% serum was added to 100. Mu.l of mannan-agarose and incubated for 30 minutes with gentle shaking or rotation at Room Temperature (RT). EDTA was then added to a final concentration of 10mM and incubated for an additional 2 minutes. Agarose was then centrifuged and the activated serum was aspirated and stored at-20 ℃.

(iii) Serial dilutions ranging from 0.1% to 20% were prepared from activated control serum and were prepared at TBS/Ca ²⁺ Is a kind of medium. The blocking buffer was discarded and 100 ul/well of sample or control serum was added to the plate and incubated for 1 hour at room temperature. The plates were then incubated with 280. Mu.l TBS/Ca ²⁺ 0.05% Tween 20 (washing buffer) 3 times

(iv) 100 μl/well of anti-C1-INH detection antibody (affinity purified rabbit polyclonal anti-C1-INH, proteintech catalog number 12259-1-AP, in TBS/Ca) was added ²⁺ Diluted 1:2000) and incubated for 1 hour at room temperature.

(v) The plates were then washed 3 times as described above. Add 100. Mu.l/Kong Kangtu HRP (Sigma, 1:5000) and incubate for an additional 45 minutes.

(vi) The plates were then washed 3 times as described above and 100 μl/well TMB substrate was added. When blue color was generated, 50. Mu.l/well 2N H was added ₂ SO ₄ To terminate the reaction and at OD _450nm And (5) measuring.

Results:

FIG. 59 graphically illustrates an OD-based ₄₅₀ The amount of MASP-2/C1-INH complex detected with each of the four candidate anti-MASP-2 mAbs (clones C1, C7, D8 and H1) at various concentrations of activated serum. Notably, the amount of MASP-2/C1-INH complex in normal, non-activated serum will be basalLines (data not shown).

As shown in FIG. 59, mAb #C7 is far superior to other anti-MASP-2 antibodies tested as capture antibodies for MASP-2/C1-INH in ELISA assays. As shown in figure 59, mAb #7 can detect MASP-2/CI-INH complexes with a dilution range of less than 5% (i.e., 0.3% to 5%) in activated human serum. Notably, the commercial antibodies from Hycult (clone 8B5 and clone 6G 12) were also tested as candidate capture antibodies in this assay format, and the results were similar to mAb C1 and D8 (i.e., not used in a sensitive ELISA assay). mAb #c7 was selected for use in highly sensitive ELISA assays and is described below.

anti-MASP-2 mAb#C7: (CDR belt underlined based on Kabat numbering System)

Heavy chain variable region：(SEQ ID NO:87)

Light chain variable region：(SEQ ID NO:88)

anti-MASP-2 mAb#C7 CDRs

#C7_VH(SEQ ID NO：95)A

#C7_VK(SEQ ID NO：96)

2. Measurement of MASP-2/C1-INH complexes in serum samples obtained from subjects in the longitudinal COVID-19 study

A subject: as described in example 24, a study was conducted to measure complement activation in subjects of various classes, in which longitudinal plasma and serum samples were taken from donors of various classes as follows:

Category:

(3) Uninfected Health Care Workers (HCW) (i.e., SARS-CoV-2 seronegative)

The assay described below uses anti-MASP-2 mAb#C7 immobilized on a microtiter plate as described above to capture MASP-2/C1-INH complexes from human serum or plasma and to detect anti-C1-INH antibodies of the captured complexes. The positive control for this assay can be prepared by incubating Normal Human Serum (NHS) with mannan-agarose, artificially activating LP and releasing MASP-2/C1-INH into the sample. Serial dilutions of the positive control can be used as calibrator/reference standard for ELISA assays. Naturally activated serum may also be used as a calibrator/reference standard, for example from a group of covd-19 patients or other patient groups known to have MASP-2 activated.

Method：

(i) Nunc Maxisorb microtiter plates with carbonate buffer (15 mM Na ₂ CO ₃ 、35mM NaHCO ₃ 100. Mu.l of anti-MASP-2 capture antibody clone #C7 (2. Mu.g/ml) in pH 9.6) was coated overnight at 4 ℃. The microtiter plates were incubated with 1% (w.sub.1) of 280. Mu.l/well TBS bufferv) BSA was blocked for 1 hour at room temperature.

(iii) From activated control serum (starting from 20%) and TBS/Ca ²⁺ Serial dilutions were prepared of the sample serum (5%). The blocking buffer was discarded and 100 ul/well of sample or control serum was added to the plate and incubated for 1 hour at room temperature. The plates were then incubated with 280. Mu.l TBS/Ca ²⁺ 0.05% Tween 20 (washing buffer) 3 times

Results:

figure 60 graphically illustrates the results of ELISA assays measuring MASP-2/C1-INH complexes in 5% serum from acute covd patients (16 samples from 3 patients <14 days after hospitalization), convalescent patients (n=15), seropositive persons (n=15), and seronegative persons (n=34). The results show the activation of the lectin pathway, measured as the amount of MASP-2/C1-INH complex, as a percentage seen in artificially activated control serum. As shown in figure 60, significantly higher amounts of MASP-2/C1-INH complex (2-3 fold higher) were observed in the serum of acute covd-19 patients (day <14 post-admission) compared to convalescent patients, seropositive and seronegative persons (analyzed with Dunnett's post-ANOVA analysis, p < 0.0001).

FIG. 61 graphically illustrates the amount of MASP-2/C1-INH complex present in 3 acute COVID-19 patients (# 2, #3, and # 4) after admission and for a period of up to 14 days after admission. The red line at the bottom of the plot shows the amount of MASP-2/C1-INH detected in the pooled normal seronegative health care personnel.

As described above, activation of the Lectin Pathway (LP) results in the formation of liquid phase MASP-2/C1-INH complexes. As shown in FIGS. 60 and 61, the LP activation of acute COVID-19 patients remained high 14-15 days after admission.

Example 26

Bead-based immunoassays for the measurement of MASP-2/C1-INH and C1s/C1-INH complexes

Background/principle:

the complement system serine protease C1s and the mannan-binding lectin associated serine protease-2 (MASP-2) circulate in the plasma as zymogens. C1s together with another serine protease C1r and recognition component C1q are part of the Classical Pathway (CP) C1 complex. MASP-2 is associated with one of several Lectin Pathway (LP) pattern recognition molecules. In both cases, the zymogen form binds loosely to the serine protease inhibitor C1-INH. When CP or LP is activated, the zymogen is cleaved into two disulfide-linked chains. Cleaved C1s and MASP-2 are active forms of enzymes that cleave downstream complement components C4 and C2. Activated MASP-2 and C1s are regulated by C1-INH, which forms a stable 1:1 complex with serine proteases. Thus, the levels of the C1s/C1-INH complex and the MASP-2/C1-INH complex in the sample provide a clear measure of recent CP or LP activation, respectively.

As described in example 25, in ELISA assays measuring MASP-2/C1-INH complex levels in 5% of serum from acute COVID patients, convalescent patients, seropositive and seronegative persons, it was determined that significantly higher amounts of MASP-2/C1-INH complex (2-3 fold higher) were observed in serum from acute COVID-19 patients (day <14 post-admission) than from convalescent patients, seropositive and seronegative persons (p <0.0001 by Dunnett's post-ANOVA analysis).

This example describes the development of a sensitive assay suitable for screening individual patient samples for the presence and/or amount of MASP-2/C1-INH complex and C1s/C1-INH complex at serum concentrations below 10% (i.e., 1% to 5%) involving the use of a Luminex platform to shift and multiplex sandwich assays into a bead-based fluorescent format.

Method：

This example provides a further analysis of MASP-2/C1-INH complex levels and analysis of C1s/C1-INH complex levels in samples from acute COVID-19 patients, convalescent patients, seropositive and seronegative persons using a high throughput bead-based immunofluorescence assay using the Luminex xMAP (Multi-analyte Profile analysis) technique.

Luminex assay of MASP-2/C1-INH and C1s/C1-INH complexes

As described herein, MASP-2/C1-INH and C1s/C1-INH complexes are specific biomarkers of lectin and classical pathway activation, respectively. To measure these biomarkers, we designed a multiplex bead based fluorescent sandwich assay, where the capture antibody (bound to the bead) is directed against serine protease and the detection antibody is directed against C1-INH.

As shown in fig. 62, a multiplex bead-based immunofluorescence assay uses anti-C1 s antibodies or anti-MASP-2 antibodies immobilized on polystyrene or magnetic polystyrene microspheres (i.e., beads) to capture serine protease/C1-INH complexes (i.e., analytes) from human serum or plasma, and anti-C1-INH antibodies as detection antibodies to detect the captured complexes.

Although the assay described in this example is based on Luminex xMAP (multi-analyte profiling) technology, those skilled in the art will appreciate that alternative bead-based immunofluorescence assays may be used to practice the claimed invention.

Bead-based assays can be multiplexed by coating one set of fluorescent beads with anti-MASP-2 monoclonal antibodies (mAbs) and coating another set of fluorescent beads with different fluorescence spectra with anti-C1 s monoclonal antibodies (mAbs).

Positive control reference standards for MASP-2/C1-INH complexes were prepared using pooled standard serum or plasma from acute covd-19 patients, as shown in the standard curve presented in figure 63 for detection of MASP-2/C1-INH complexes with mAb C8 anti-MASP-2 as capture antibody. As shown in fig. 63, the bead-based assay is capable of detecting MASP-2/C1-INH complexes in less than 10% plasma or serum (i.e., 0.1% to 10% plasma or serum, such as 0.5% to 8%, or 0.5% to 7.5%, such as 1% to 5% serum or plasma) from an acute covd-19 patient.

Alternatively, positive controls for MASP-2/C1-INH complexes can be prepared by incubating normal human serum with mannan agarose, artificially activating the Lectin Pathway (LP), and releasing the MASP-2/C1-INH complex into the sample. Likewise, positive controls for the C1s/C1-INH complex can be prepared by incubating normal human serum with the immune complex, artificially activating the CP, and releasing the C1s/C1-INH complex into the sample. Serial dilutions of positive controls can be used as calibrators.

As another alternative, the standard and positive control can be prepared by: stoichiometric amounts of recombinant C1-INH were mixed with recombinant C1s or recombinant MASP-2, and the resulting C1s/C1-INH or MASP-2/C1-INH complex was purified by size exclusion chromatography and quantified by gel electrophoresis and/or Bradford determination or measurement of absorbance at 280nm, as further described in example 27.

Bead-based assay：

Antibody coated magnetic beads: a panel of monoclonal antibodies known to bind MASP-2 and C1s was tested as capture antibodies. Antibodies were diluted to 50 μg/ml in Phosphate Buffered Saline (PBS) and immobilized on MagPlex magnetic polystyrene microspheres (Luminex) by carbodiimide coupling using xMAP antibody coupling kit as described in Luminex (xMAP) cookie (4 th edition). After coupling, any remaining reaction sites on the beads were blocked by incubation with PBS containing 0.05% TWEEN 20, pH 7.4 (PBS TBN). BSA-coated beads were prepared as negative controls. Magplex beads with different emission spectra were used for anti-C1 s, anti-MASP-2 and BSA coated beads to allow multiplexing of assays.

Measurement procedure: the antibodies and BSA-coupled MagPlax beads were diluted in PBS-TBN assay buffer to a final concentration of 50 beads/. Mu.L for each type of bead. mu.L of this mixture was aliquoted into each well of a 96-well plate. Equal volumes of plasma or serum diluted in PBS-TBN were added to the wells, mixed and incubated on a shaker for 30 minutes at room temperature. The beads were washed 3 times with assay buffer as follows: the beads were retained with a magnetic separator, the supernatant was aspirated and 100. Mu.L of fresh PBS-TBN assay buffer was added. After washing, the beads were resuspended in 50. Mu.L assay buffer and bound ligand was detected by addition of biotinylated anti-C1-INH polyclonal antibody (R & D Systems, BAF 2488) at a 1:1000 dilution in PBS-TBN assay buffer. The beads were incubated with the detection antibody for 30 minutes at room temperature and then washed 3 times as described above. Streptavidin R-phycoerythrin (SAFE; thermo Fisher Scientific) was diluted to 1. Mu.g/mL in assay buffer, 100. Mu.L was added to each well, mixed and incubated for 30 min at room temperature. After washing as described above, 50-75. Mu.L of each reaction was analyzed on a Luminex analyzer according to the System handbook.

Antibody selection: in a preliminary experiment designed to test capture and detection antibody pairs, we prepared control serum for MASP-2/C1-INH assays by: normal human serum was incubated with mannan-agarose, LP was activated artificially and MASP-2/C1-INH was released into the sample. Likewise, a positive control for the C1s/C1-INH complex was prepared by incubating normal human serum with sheep anti-HSA, generating an immune complex in situ to artificially activate CP and release C1s/C1-INH into the sample. Serial dilutions of these sera ranging from 1:10 to 1:1280 were assayed as described above. The controls were: unactivated NHS, BSA-coated beads, serum-free (buffer only) reaction, and omission of the detection antibody mixture. The following mabs were shown to function well as capture abs.

anti-MASP-2 humanized mouse mAb #C8

anti-C1 s affinity purified polyclonal, proteintech (14554-1-AP)

These capture antibodies give a simple log/linear relationship between sample concentration and fluorescence intensity at sample dilutions of 1:10 to 1:640, with the signal falling to background levels at 1:1280. The anti-MASP-2 mAb clone 8B5 (Hycult Biotech) previously used successfully in sandwich ELISA performed poorly in the Luminex assay, was poorly sensitive and had a low signal to noise ratio.

Exemplary assay protocol

(i) 250. Mu.L of polystyrene or magnetic polystyrene microbeads (e.g., mag. Mu. Lex beads) were coated with 12.5. Mu.g of capture antibody in 250. Mu.L of phosphate buffered saline using the xMAP antibody coupling kit according to the manufacturer's instructions (see Luminex) (xMAP version 4). The following monoclonal antibodies have been shown to function as capture antibodies.

anti-MASP-2 Capture antibody clone #C8

anti-C1 s affinity purified polyclonal, catalog number 14554-1-AP, proteintech

The capture antibodies should be conjugated to different sets of beads, one for each antibody.

(ii) To prepare an activated control serum, pooled normal human serum was diluted to 5mM Ca ²⁺ 20% v/v Tris-buffered saline (TBS; 10mM Tris-Cl,140mM NaCl, pH 7.4). Mu.l of mannan-agarose (Sigma catalog number M9917) was treated with 5 volumes of TBS/Ca ²⁺ Washed twice and resuspended to 100 μl in the same buffer. mu.L of 20% serum was added to 100. Mu.L of mannan-agarose and incubated for 30 minutes with gentle shaking or rotation at Room Temperature (RT). EDTA was added to a final concentration of 10mM and incubated for an additional 2 minutes, after which the agarose was centrifuged and the activated serum was aspirated. Stored at-20 ℃.

(iii) Appropriate sets of antibody-coupled microspheres were selected and assayed as follows:

Resuspending the microspheres by vortexing and sonication

The working microsphere mixture was prepared by diluting the coupled microsphere stock in assay buffer (TBS) to a final concentration of 50 microspheres/μl per group.

Aliquoting 50 μl of the working microsphere mixture into wells of an appropriate 96-well plate.

Add 50 μl of assay buffer (TBS) to each background well.

Add 50 μl of standard or sample to the appropriate wells.

Gently mix the reactants by pipetting up and down several times with a multichannel pipettor.

Cover plate and incubate on a shaker set at about 800rpm for 30 minutes at room temperature.

Place the plate in a magnetic separator and allow separation to occur for 30-60 seconds.

Carefully aspirate supernatant from each well using a multichannel pipettor.

Leave the plate in the magnetic separator for the following washing steps:

100 μl of assay buffer was added to each well.

The supernatant was carefully aspirated from each well using a multichannel pipettor

The plate was removed from the magnetic separator and the microspheres were resuspended in 50 μl of assay buffer by gently pipetting up and down several times using a multichannel pipette.

Diluting the biotinylated detection antibody in assay buffer. Suitable detection antibodies are polyclonal antibodies affinity purified by the R & D system against C1-INH, catalog number BAF2488, diluted 1:1000.

Add 50 μl of diluted detection antibody to each well.

Cover plate and incubate on a plate shaker set at about 800rpm for 30 minutes at room temperature.

Carefully aspirate supernatant from each well using a multichannel pipettor.

Leave the plate in the magnetic separator for the following washing steps:

100 μl of assay buffer was added to each well.

Streptavidin, R-phycoerythrin conjugate (SAFE) reporter was diluted to 1 μg/mL in detection buffer.

Add 50. Mu.L of diluted SAFE to each well.

Carefully aspirate supernatant from each well using a multichannel pipettor.

Leave the plate in the magnetic separator for the following washing steps:

100 μl of assay buffer was added to each well.

The plate was removed from the magnetic separator and the microspheres were resuspended in 100 μl of assay buffer by gently pipetting up and down several times with a multichannel pipette.

50-75. Mu.L was analyzed on a Luminex analyzer according to the System handbook.

anti-MASP-2 mAb#C8

mAb#C8 VH(SEQ ID NO：97)

mAb#C8 VH(SEQ ID NO：98)

Results：

FIG. 63 graphically illustrates the detection of MASP-2/C1-INH complex in pooled human serum from acute COVID-19 patients in a bead-based assay using anti-MASP-2 mAb#C8 as capture antibody, as compared to BSA coated control beads.

Example 27

Methods of generating MASP-2/C1-INH complexes for use as reference standards

Method：

1. Human MASP-2 CCP1/CCP2/SP6His (MW 43,740) was mixed with a C1 esterase inhibitor (Sigma E0518 (from human plasma) MW 105,000 in a molar ratio of MASP-2: C1 inhibitor 1:1.5 (300. Mu.g total).

2. Shaking in an eppendorf tube at 400rpm for 60 minutes, 37℃C

3. Refrigerating overnight

4. Purification by Size Exclusion Chromatography (SEC):

Table 16: SEC analysis and purification

Peak to peak	Reservation of	Area percent
			1	8.478	56.09
2	10.588	40.6
			3	13.242	3.31

Peak 1 and Peak 2 were collected from SEC and run on non-reducing gels along with control MASP-2 CCP1/CCP2/SP6HIS (MW 43,740) and C1 esterase inhibitor (MW 105,000).

Results：

FIG. 64 is a photograph of a non-reducing gel loaded with 6. Mu.g of sample obtained during SEC purification of recombinant MASP-2/C1-INH complex, wherein: lane 1: peak 1 flow-through; lane 2: peak 1 concentrate; lane 3: peak 2 flow-through; lane 4: peak 2 concentrate; lane 5: an unpurified mixture; lane 6: MASP-2 CCP1/2/SP (43,740KD); lane 7: c1-inhibitor (100 KD).

As shown in FIG. 64, the purified MASP-2/C1-INH complex is present in the concentration peak 1. The recombinant complexes can be used as reference standards in bead-based assays as described in example 26.

Example 28

Acute COVID-19 patients tested for levels of MASP-2/C1-INH and C1s/CI-INH complexes

Method：

A study was performed to measure complement activation in longitudinal plasma and serum samples taken from various types of COVID-19 patients and healthy volunteers as described in examples 24 and 25. As described herein, acute covd-19 infection results in complement activation, decompensation, and release of complement activation products (e.g., C5 a).

As part of this ongoing longitudinal study, 40 patients with severe acute COVID-19 were tested for MASP-2/C1-INH complex formation and C1s/C1-INH complex formation using the bead-based assay described in example 26, as described in this example.

Forty (40) patients with severe acute covd-19 analyzed in this example were enrolled in the Royal Papworth hospital (Royal Papworth Hospital, UK) united kingdom. WHO clinical scores for these patients ranged from 3-7, with 19 requiring in vitro epicardial lung oxygenation (ECMO). 19 of the patients survived and were recalled for follow-up at and 3 months after discharge; 21 people die from the disease. 30 common Health Care Workers (HCW) tested positive but not hospitalized with the COVID-19 and 30 uninfected HCW served as controls.

Plasma samples taken from each subject at the indicated times (shown as time to admission) were diluted 1:50 and analyzed for levels of MASP-2/C1-INH complex and C1s/C1-INH complex in a bead-based dual Luminex assay, as described in example 26.

Measurement of complement hemolysis (CH 50)

Antibody-driven sheep red blood cell (SE) complement cleavage was measured using rabbit anti-sheep IgG coated SE as follows. Sheep red blood cells (Oxoid) were washed 3 times with GVB buffer (10 mM barbital, 145mM NaCl, 0.1% w/v gelatin) containing 10mM EDTA. The final concentration of RBCs was adjusted to 1x10 ⁹ /ml. RBCs were sensitized by gentle shaking incubation with anti-sheep RBCs (Sigma S1389,1:200 dilution) for 30 minutes at 37 ℃. Finally, using a solution containing 2mM Ca ²⁺ And 1mM Mg ²⁺ GVB buffer (GVB) ⁺⁺ ) RBCs were washed. 100. Mu.l GVB of serum samples in 96-well plates ⁺⁺ Serial dilutions in buffer and equal volumes of GVB ⁺⁺ 10 in (2) ⁷ Individual RBCs were added to each well. Wells containing buffer only served as negative controls. Wells containing water instead of buffer/plasma were used as positive controls (typically 100% cut). After incubation for 30 minutes at 37 ℃, the plates were centrifuged, 100 μl of supernatant was aspirated and the released hemoglobin was determined by measuring OD at 405 nm. Percent hemolysis was calculated and plotted against plasma dilution to determine CH50.

Circulation C5a (cat No. DY 2037) was measured using a proprietary sandwich ELISA provided by the R & D system.

Results：

FIG. 65 graphically illustrates the level of MASP-2/C1-INH complex in acute COVID-19 patients as determined in the dual bead-based assays described herein. As shown in FIG. 65, MASP-2/C1-INH complex levels were elevated throughout the acute phase of the disease compared to healthy controls, indicating lectin pathway activation in acute COVID-19. As further shown in FIG. 65, three months after discharge, reduced but abnormal levels of MASP-2/C1-INH complex were observed in survivors. In contrast, persons with mild covd-19 disease (seropositive Health Care Workers (HCW)) showed no elevation in MASP-2/C1-INH levels. Hospitalized patient n=40, non-hospitalized covd-19 case n=30, healthy control n=30. Multiple comparisons were made by one-way ANOVA analysis with Dunnett's correction.

FIG. 66 graphically illustrates the level of C1s/C1-INH complex in acute COVID-19 patients as determined in the dual bead-based assays described herein. As shown in FIG. 66, the C1s/C1-INH complex levels were elevated during the acute phase of the entire disease compared to healthy controls, indicating classical pathway activation in acute COVID-19. As further shown in fig. 66, a decrease in C1s/C1-INH complex levels was observed in survivors, but not normal, three months after discharge. In contrast, humans with mild covd-19 disease (seropositive Health Care Workers (HCW)) showed no elevation in C1s/C1-INH levels. Hospitalized patient n=40, non-hospitalized covd-19 case n=30, healthy control n=30. Multiple comparisons were made by one-way ANOVA analysis with Dunnett's correction. Notably, the C1s/C1inh complex levels correlated with anti-COVID-19 antibody titers and Antibody Dependent Complement Deposition (ADCD) (data not shown).

FIG. 67 graphically illustrates CH50 values for acute COVID-19 patients, convalescent patients, seropositive persons, and seronegative persons in the longitudinal study described in this example. As shown in fig. 67, CH in acute phase of disease compared to healthy controls ₅₀ Lower values indicate complement consumption and activation.

FIG. 68 graphically illustrates the C5a values for acute COVID-19 patients, convalescent patients, seropositive and seronegative persons in the longitudinal study described in this example. As shown in fig. 68, the C5a value was higher in the acute phase of the disease compared to healthy controls, indicating complement activation.

Taken together, the results indicate that complement consumption and activation occur in the early acute phase of covd-19 even in the absence of anti-covd-19 antibodies.

As shown in FIGS. 65 and 66, levels of MASP-2/C1-INH complex and C1s/C1-INH complex were significantly elevated in all hospitalized acute COVID-19 patients. In those surviving, the serine protease/C1-INH complex levels tended to be normal three months after discharge, although the levels in a fraction of the patients still increased, making the values higher than the control group, and indicating ongoing complement activation.

The measurement performance: SUMMARY

As described herein, acute covd-19 infection results in complement activation, decompensation, and release of complement activation products such as C3a and C5 a. We tested a bead-based C1-INH complex assay using plasma from 40 patients with severe acute covd-19, enrolled in Royal Papworth hospital, england. WHO clinical scores for these patients ranged from 3-7, and 19 of them required in vitro epicardial lung oxygenation (ECMO). 19 of the patients survived; 21 people die from the disease. 30 uninfected Health Care Workers (HCW) served as controls. Serial dilutions of pooled acute phase plasma were used as standard (range 1:10-1:1280). Each sample was diluted 1:50 in assay buffer. High and low standards (combined acute and NHS) were included at 3 different locations on each plate to determine intra-and inter-plate variability.

Both standard curves were linear log/linear, with available plasma dilutions ranging from 1:20 to 1:640. The absolute fluorescence signal of the C1s/C1-INH complex is about 10-fold higher than that of the MASP-2/C1-INH complex, possibly reflecting the difference in serum concentration between MASP-2 and C1 s.

MASP-2/C1-INH complex and C1s/C1-INH complex were significantly elevated in all hospitalized acute COVID-19 patients compared to healthy controls, indicating activation of both LP and CP.

Example 29

Anti-treatment with naloxone Li Shan reduced the level of MASP-2/C1-INH complex in COVID-19 and resulted in better clinical results.

Background/principle:

as described in examples 20, 21 and 22, the lectin pathway has been shown to cause lung injury associated with acute COVID-19, and the representative MASP-2 inhibitory antibody, nano-Li Shan, is effective in alleviating pulmonary symptoms in patients with acute COVID-19. In this example, acute COVID-19 patients treated with naloxone Li Shan as described in examples 20, 21 and 22 were analyzed to determine the effect of naloxone Li Shan on MASP-2/C1-INH complex levels.

Method：

8 patients with acute COVID-19 were sent to the ITU of Italy Bergamo and treated with Nasoh Li Shan at a dose of 4mg/kg twice a week. Samples were collected at admission (prior to treatment with naloxone Li Shan) and then calculated for days after treatment with naloxone Li Shan, samples were collected on days 3-4, 7-8 and 9 to discharge. 16 healthy control subjects were enrolled simultaneously.

Samples were analyzed for CH50, C5a and MASP-2/C1-INH complexes using the bead-based assay described in example 26.

Results：

FIG. 69 graphically illustrates the levels of MASP-2/C1-INH complex in samples from 8 acute COVID-19 patients at admission (before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9 to discharge) as compared to 16 healthy controls. As shown in FIG. 69, MASP-2/C1-INH complex levels were elevated in acute COVID patients at the time of admission (prior to nasucasian Li Shan anti-treatment) compared to healthy subjects. As further shown in figure 69, MASP-2/C1-INH complex levels were drastically reduced on days 3-4 after the nano-cable Li Shan anti-treatment compared to levels observed in healthy controls, which continued until discharge. In contrast, in acute covd-19 patients untreated with naloxone Li Shan from the longitudinal study described in example 28 as shown in fig. 65, elevated levels of MASP-2/C1-INH complex were observed on days 0-4, 5-10, and 11 to discharge, with reduced levels of MASP-2/C1-INH complex occurring at 3 months of follow-up, which was still higher than the normal healthy control.

FIG. 70A graphically illustrates CH in samples from 8 acute COVID-19 patients at admission (before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9-discharge) compared to 16 healthy controls ₅₀ Values.

As shown in FIG. 70A, the CH of an acute COVID-19 patient at the time of admission (prior to naloxone Li Shan anti-treatment) ₅₀ The values were lower than healthy controls. As further shown in fig. 70A, CH was on days 3-4 after the naloxone Li Shan anti-treatment ₅₀ To the normal range, which continues to increase by day 7-8 and remains within the normal range from day 9 to discharge. In contrast, CH was observed on days 0-4 in acute covd-19 patients untreated with nano-cable Li Shan as shown in fig. 67 from the longitudinal study described in example 28, compared to seronegative personnel ₅₀ The values decreased and the lower levels continued to day 5-10 and day 11-15 and eventually increased to normal levels at 3 months follow-up in convalescent patients.

Figure 70B graphically illustrates the C5a values in samples from 8 acute covd-19 patients at admission (prior to naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 3-4 after initiation of treatment; days 7-8, 9 to discharge) compared to 16 healthy controls.

As shown in figure 70B, the C5a value of acute covd-19 patients at admission (prior to nasucasian Li Shan anti-treatment) was significantly higher than healthy control subjects. As further shown in fig. 70B, the C5a value drops sharply at days 3-4 after the nano-cable Li Shan anti-treatment and continues to drop to near normal levels at days 7-8 and 9 to discharge. In contrast, in acute covd-19 patients untreated with naloxone Li Shan as shown in fig. 68 from the longitudinal study described in example 28, a significant increase in C5a values was observed on days 0-4, which decreased over time, but remained higher than normal on days 5-10 and 11-15, and then eventually decreased to normal levels by 3 months of follow-up (convalescent patients), as compared to seronegative personnel.

Taken together, these results indicate that patients with acute covd-19 have complement activation and consumption at admission, and that anti-treatment with nano-cable Li Shan rapidly reduces complement activation and consumption. It was further shown that MASP-2/C1-INH complex levels indicate complement activation status in patients with COVID-19, which was found to be high at hospitalization and rapidly declined following anti-treatment with naloxone Li Shan. Thus, the level of MASP-2/C1-INH complex can be used as a means of determining the need for anti-treatment with naloxone Li Shan, and can also be used to monitor the efficacy of the naloxone Li Shan anti-treatment.

Discussion:

as described in this example, we examined the effect of naloxone Li Shan anti-treatment on complement activation during acute COVID-19. Markers of complement activation and depletion were analyzed in longitudinal plasma samples taken from 8 patients hospitalized for acute Covid-19 (WHO score 3-7). Samples taken from healthy healthcare workers served as controls.

All patients were anticomplemented immediately prior to treatment (low CH ₅₀ ) And shows evidence of alternative pathway activation and anaphylatoxin production (Bb and C5a production). Using the novel bead-based fluorescent immunoassays described herein to measure the C1s/C1-INH and MASP-2/C1-INH complexes (specific markers of classical pathway and lectin pathway activation, respectively), we found that the levels of both complexes were significantly elevated in all patients prior to treatment. The nano-cable Li Shan anti-treatment resulted in a rapid and sustained decrease in MASP-2/C1Inh complex and a corresponding decrease in C5a production. The C1s/C1Inh levels remained high throughout the acute phase.

Together with previous clinical results, these findings suggest that anti-targeting of the lectin pathway with nano-wire Li Shan may be sufficient to reduce complement activation and anaphylatoxin to below the threshold to maintain ARDS, even in the presence of ongoing activation of the classical pathway.

These data indicate that lectin pathway activation is very high very early in severe covd-19. This excessive lectin pathway activation causes consumption of complement components shared between the lectin pathway and the classical pathway, thereby compromising classical pathway function. Evaluation of blood samples from the Bergamo test showed that inhibition of the lectin pathway by the nano-cable Li Shan antibody can restore loss of classical pathway functional activity caused by uncontrolled consumption by excessive activation of the lectin pathway. These results indicate that the MASP-2/C1-INH complex can be used as an early indicator of severe COVID-19 and as a means of assessing therapeutic response in a patient with COVID-19 undergoing treatment.

Example 30

Further evidence that anti-treatment with naloxone Li Shan reduced the level of MASP-2/C1-INH complex in COVID-19 and led to better clinical outcome was demonstrated.

Background/principle:

as described in examples 20, 21, 22 and 23, the lectin pathway has been shown to cause lung injury associated with acute COVID-19, and the representative MASP-2 inhibitory antibody, nasoh Li Shan, is effective in alleviating pulmonary symptoms in patients with acute COVID-19. In this example, patients with acute covd-19 treated with naloxone Li Shan as described in examples 20, 21 and 22 were analyzed to determine the effect of naloxone Li Shan resistance on MASP-2/C1-INH complex levels. Patients with acute covd-19 were determined to have complement activation and consumption at admission and anti-treatment with naloxone Li Shan rapidly reduced complement activation and consumption as described in example 29. It was further demonstrated that MASP-2/C1-INH complex levels indicate complement activation status in patients with COVID-19, which was found to be high at hospitalization and decreased rapidly after anti-treatment with naloxone Li Shan. This example describes a further analysis of longitudinal samples obtained from acute subjects with acute covd-19 treated with naloxone Li Shan (treatment group) compared to longitudinal samples obtained from subjects with acute covd-19 treated with no naloxone Li Shan treated with naloxone Li Shan (untreated group) who received treatment during the same period of time, wherein the samples were analyzed for CH50, C5a and MASP-2/C1-INH complexes using the bead-based immunoassay as described in example 26.

Method：

Patients and controls included in the study

We analyzed longitudinal plasma samples from 16 moderate and severe patients with covd-19; 7 were treated with naloxone Li Shan (with all recovered and discharged after treatment) and 9 were untreated, all admitted to the ICU of Papa Giovanni XXIII hospital of Bergamo, italy, in the fourth quarter of 2020. All patients were positive for SARS-CoV-2PCR, had ARDS (according to Berlin standard (Ferguson N.D. et al Intensive Care Med (10): 1573-82, 2012)), required at least CPAP (sustained passive airway pressure (continuous passive airway pressure)). The most ill patients were selected for anti-treatment with Naline Li Shan. All patients received enoxaparin, dexamethasone and 500mg azithromycin daily, but patients with active systemic bacterial or fungal infection who required further antimicrobial therapy were not suitable for anti-treatment with Naline Li Shan.

In the treatment cohort, naloxone Li Shan antibody (4 mg/kg) was administered intravenously twice weekly for 2-4 weeks. Blood was collected prior to each dose of nano-cord Li Shan antibody, and then twice weekly. Citrate plasma was prepared and frozen at-80 ℃ until analysis. In parallel, samples were collected from COVID-19 patients who did not receive the resistance to Naxol Li Shan. Also, normal control plasma was collected from 17 seronegative volunteers (health care workers). Samples were analyzed for CH50, C5a and MASP-2/C1-INH complexes using the bead-based assay described in example 26.

Complement Hemolysis (CH) ₅₀ ) Is measured by (a)

C5a ELISA

Circulation C5a was measured using a proprietary sandwich ELISA provided by the R & D system (accession number DY 2037).

Serum Bactericidal Assay (SBA)

The klebsiella pneumoniae isolate was grown overnight with gentle shaking in a nutrient broth at 37 ℃. The next day, 10mL of fresh nutrient broth was inoculated with 100 μl of overnight bacterial culture and incubated at 37 ℃ with gentle shaking until mid-log phase. Bacterial cultures were collected using BBS (4 mM barbital, 145mM NaCl, 2mM CaCl2, 1mM MgCl) ₂ pH 7.4) was washed twice and then adjusted to 1X 10 ⁷ Final concentration of CFU mL-1. Will be 1X 10 ⁵ CFU were incubated with 50% serum from HCW or serum from acute covd-19 patients before and after nano-cable Li Shan anti-treatment in BBS with gentle shaking at 37 ℃. After 2 hours, samples were taken and spread on nutrient agar plates at 37 ℃ overnight. Serum from patients was incubated with klebsiella pneumoniae (ATCC 43816) for 120 minutes and recoverable viable bacterial colonies were calculated by measuring the decrease in viable bacterial count recovered after 2 hours incubation with each serum compared to heat-inactivated normal human serum (HI-NHS).

Luminex assay of MASP-2/C1Inh and C1s/C1Inh complexes

MASP-2/C1-INH and C1s/C1-INH complexes are specific markers of lectin and classical pathway activation, respectively. To measure these markers we used a multiplex bead-based fluorescent sandwich assay as described in example 26.

Results:

in this study, seven severe cases of covd-19 were treated with naloxone Li Shan antibody (final concentration of 4mg/kg body weight administered intravenously twice weekly), all of which recovered after treatment. For comparison, longitudinal blood samples were collected from a control group (untreated group) of 9 covd-19 patients who received no resistance to nascidin Li Shan in the ICU during the same time period.

Naloxone Li Shan anti-treatment reduced the highly elevated lectin pathway activation levels in covd-19 patients to the levels seen in healthy controls.

We used the bead-based fluorescence assay described in example 26, which we developed to monitor the activation status of the Lectin Pathway (LP) and Classical Pathway (CP) by detecting MASP-2/C1-INH and C1s/C1-INH complexes in serum/plasma of patients with COVID-19.

Figure 71 graphically illustrates the levels of MASP-2C1-INH complex in samples from 7 covd-19 patients at admission (day 0, before naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 2-4 after initiation of treatment; days 6-8 and 9 to discharge) compared to samples obtained from 9 covd-19 patients untreated with naloxone Li Shan anti-treatment (untreated control) and a group of healthy control subjects (healthy control) during the same period. As shown in FIG. 71, at the beginning of the study, all patients had high levels of MASP-2/C1-INH complex, indicating lectin pathway activation. Naloxone Li Shan anti-treatment reduced MASP-2/C1-INH to normal levels immediately after the first administration. Levels of MASP-2/C1-INH complex in the untreated group were significantly higher than those in the treated group for the remainder of the study (p < 0.001). Results were analyzed using one-way ANOVA. In contrast, the resistance of nano-cable Li Shan had no effect on the generation of classical pathway driven C1s/C1-INH complex, which remained high in both patient groups throughout the study (data not shown).

Naloxone Li Shan anti-treatment ameliorates hypocomplement in acute covd 19

FIG. 72A graphically illustrates CH in samples from 7 acute COVID-19 patients at admission (day 0, prior to naloxone Li Shan treatment) and after naloxone Li Shan treatment (days 2-4 after initiation of treatment; days 6-8 and 9 to discharge) compared to samples obtained from 9 acute COVID-19 patients untreated with naloxone Li Shan during the same time period (untreated control) and a group of healthy control subjects (healthy control) ₅₀ Values.

Figure 72B graphically illustrates C5a values in samples from 7 acute covd-19 patients at admission (day 0, prior to naloxone Li Shan anti-treatment) and after naloxone Li Shan anti-treatment (days 2-4 after initiation of treatment; days 6-8 and 9 to discharge) compared to samples obtained from 9 acute covd-19 patients untreated with naloxone Li Shan anti-treatment (untreated control) and a group of healthy control subjects (healthy control) during the same period.

As shown in FIG. 72A, at the beginning of the study, plasma from all 16 COVID-19 patients had as low CH ₅₀ Severe hypocomplement was determined by the values. As further shown in fig. 72A, nano-cord Li Shan anti-treatment resulted in CH ₅₀ Immediate improvement in the values indicates restoration of complement activation. In contrast, untreated control covd-19 patients had significantly lower CH during the first 9 days of the study ₅₀ Value (p=0.0093), and normal complement activation is resumed only shortly before discharge. As shown in FIG. 72B, upon entering the ICU, all COVID-19 patients had high levels of anaphylatoxin C5a in their serum, reflecting that complement activation had occurred. As further shown in fig. 72B, the nano-cord Li Shan anti-treatment reduced C5a plasma levels to normal levels 3 days after the first dose and remained normal for the duration of the study.

In summary, in the naloxone Li Shan anti-treatment group, CH ₅₀ And C5a both returned to normal by 3 days after the first dose and remained normal for the duration of the study. In the untreated group, CH compared to the treated group in the remainder of the study ₅₀ The values were significantly lower (p=0.0093) and the C5a levels were significantly higher (p=0.023). Results were analyzed using one-way ANOVA.

Nasoh Li Shan anti-treatment inhibits lectin pathway mediated complement activation, which allows recovery of classical pathway activity and antibody mediated bactericidal activity

Complement-mediated bacterial lysis plays an important role in the defense against microbial infections, especially against gram-negative bacteria. Hypocomplement induced by severe covd-19 severely impairs serum conditioning or kills the ability of klebsiella pneumoniae (a major secondary complication in covd-19). To determine whether the naso Li Shan antibody could reverse this loss of function, we measured Serum Bactericidal Activity (SBA) against klebsiella pneumoniae in serum from treated and untreated patients.

FIG. 73 graphically illustrates the viable bacterial count of Klebsiella pneumoniae after incubation with serum from a patient with COVID-19 prior to treatment with naloxone Li Shan (pre-treatment) and a patient with COVID-19 after treatment with naloxone Li Shan, compared to serum from a patient with COVID-19 not treated with naloxone Li Shan, compared to Normal Healthy Serum (NHS) and heat-inactivated normal healthy serum (HI-NHS). As shown in fig. 73, significantly lower bacterial counts were observed when serum from patients after anti-treatment with naloxone Li Shan was used as compared to serum collected prior to treatment. The opsonization of klebsiella pneumoniae by C3b was also compromised when incubated in pooled (n=6) serum from acute covd-19 patients. After administration of the naloxone Li Shan antibody, pooled serum (n=6) from the same patient was conditioned with C3b for klebsiella pneumoniae to a similar extent as pooled normal healthy controls. As negative control, a heat-inactivated normal healthy control was used. Multiple comparisons were made using one-way ANOVA analysis results with Dunnett's correction.

General overview of the results:

as described herein and in Rambaldi A. Et al, immunobiology 225 (6): 152001,2020, treatment of critically ill patients with Naso Li Shan antibody, a MASP-2 inhibitory antibody that inhibits the Lectin Pathway (LP), achieved therapeutic breakthrough that disease manifestations improved rapidly after infusion.

In this example, we examined the effect of naloxone Li Shan anti-treatment on complement activation during acute covd-19. Markers of complement activation and depletion were analyzed in longitudinal plasma samples taken from seven patients hospitalized with acute covd-19 (WHO scores 3-7) and treated with resistance to nasosin Li Shan, all of which were restored and discharged. Samples taken from healthy healthcare workers and untreated covd-19 served as controls.

Plasma from all patients exhibited low CH prior to treatment ₅₀ And high C5a anaphylatoxin levels, which are indicative of the passage of all three complementsMarkers of complement activation of the activation pathway. Using a novel bead-based fluorescent immunoassay to measure the C1s/C1-INH and MASP-2/C1-INH complexes (specific markers of classical pathway and lectin pathway activation, respectively), we found that the levels of both complexes were significantly elevated in all patients prior to treatment. The anti-treatment with nano-cable Li Shan resulted in a rapid and sustained decrease in MASP-2/C1-INH complex and a corresponding decrease in C5a production. The C1s/C1-INH levels remained high throughout the acute phase. The bactericidal activity against gram-negative bacteria is also restored.

Together with previous clinical results, these findings provide further evidence that targeting the lectin pathway with naso Li Shan may be sufficient to reduce complement activation and anaphylatoxins below the threshold for maintaining ARDS, even in the presence of classical pathway activation ongoing, and restore SBA required for successful defense against opportunistic secondary infections.

Other embodiments

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

Various modifications and variations of the methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

While illustrative embodiments have been shown and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Sequence listing

<110> Omeros Corporation

Demopulos, Gregory A.

Dudler, Thomas

Lynch, Nicholas James

Schwaeble, Hans-Wilhelm

Shaffer, Kathleen A.

Yabuki, Munehisa

<120> biomarkers for assessing risk of developing acute covd-19 and post-acute covd-19

<130> MP.1.0319.PCT

<150> 63/146,479

<151> 2021-02-05

<150> 63/277,361

<151> 2021-11-09

<160> 98

<170> PatentIn version 3.5

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gcgccacagc ctccctgcag ggtgtggggt gggagcacag gcctgggcct caccgcccct 240

gccctgccca taggctgctg accctcctgg gccttctgtg tggctcggtg gccaccccct 300

taggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc atcccccggc tttccagggg 360

agtatgccaa tgaccaggag cggcgctgga ccctgactgc accccccggc taccgcctgc 420

gcctctactt cacccacttc gacctggagc tctcccacct ctgcgagtac gacttcgtca 480

aggtgccgtc agacgggagg gctggggttt ctcagggtcg gggggtcccc aaggagtagc 540

cagggttcag ggacacctgg gagcaggggc caggcttggc caggagggag atcaggcctg 600

ggtcttgcct tcactccctg tgacacctga ccccacagct gagctcgggg gccaaggtgc 660

tggccacgct gtgcgggcag gagagcacag acacggagcg ggcccctggc aaggacactt 720

tctactcgct gggctccagc ctggacatta ccttccgctc cgactactcc aacgagaagc 780

cgttcacggg gttcgaggcc ttctatgcag ccgagggtga gccaagaggg gtcctgcaac 840

atctcagtct gcgcagctgg ctgtgggggt aactctgtct taggccaggc agccctgcct 900

tcagtttccc cacctttccc agggcagggg agaggcctct ggcctgacat catccacaat 960

gcaaagacca aaacagccgt gacctccatt cacatgggct gagtgccaac tctgagccag 1020

ggatctgagg acagcatcgc ctcaagtgac gcagggactg gccgggcgcg gcagctcacg 1080

cctgtaattc cagcactttg ggaggccgag gctggcttga taatttgagg gtcaggagtt 1140

caaggccagc cagggcaaca cggtgaaact ctatctccac taaaactaca aaaattagct 1200

gggcgtggtg gtgcgcacct ggaatcccag ctactaggga ggctgaggca ggagaattgc 1260

ttgaacctgc gaggtggagg ctgcagtgaa cagagattgc accactacac tccacctggg 1320

cgacagacta gactccgtct caaaaaacaa aaaacaaaaa ccacgcaggg ccgagggccc 1380

atttacaagc tgacaaagtg ggccctgcca gcgggagcgc tgcaggatgt ttgattttca 1440

gatcccagtc cctgcagaga ccaactgtgt gacctctggc aagtggctca atttctctgc 1500

tccttagaag ctgctgcaag ggttcagcgc tgtagccccg ccccctgggt ttgattgact 1560

cccctcatta gctgggtgac ctcggccgga cactgaaact cccactggtt taacagaggt 1620

gatgtttgca tctttctccc agcgctgctg ggagcttgca gcgaccctag gcctgtaagg 1680

tgattggccc ggcaccagtc ccgcacccta gacaggacct aggcctcctc tgaggtccac 1740

tctgaggtca tggatctcct gggaggagtc caggctggat cccgcctctt tccctcctga 1800

cggcctgcct ggccctgcct ctcccccaga cattgacgag tgccaggtgg ccccgggaga 1860

ggcgcccacc tgcgaccacc actgccacaa ccacctgggc ggtttctact gctcctgccg 1920

cgcaggctac gtcctgcacc gtaacaagcg cacctgctca ggtgagggag gctgcctggg 1980

ccccaacgca ccctctcctg ggatacccgg ggctcctcag ggccattgct gctctgccca 2040

ggggtgcgga gggcctgggc ctggacactg ggtgcttcta ggccctgctg cctccagctc 2100

cccttctcag ccctgcttcc cctctcagca gccaggctca tcagtgccac cctgccctag 2160

cactgagact aattctaaca tcccactgtg tacctggttc cacctgggct ctgggaaccc 2220

ctcatgtagc cacgggagag tcggggtatc taccctcgtt ccttggactg ggttcctgtt 2280

ccctgcactg ggggacgggc cagtgctctg gggcgtgggc agccccaccc tgtggcgctg 2340

accctgctcc cccgactcgg tttctcctct cggggtctct ccttgcctct ctgatctctc 2400

ttccagagca gagcctctag cctcccctgg agctccggct gcccagcagg tcagaagcca 2460

gagccaggct gctggcctca gctccgggtt gggctgagat gctgtgcccc aactcccatt 2520

cacccaccat ggacccaata ataaacctgg ccccacccca cctgctgccg cgtgtctctg 2580

gggtgggagg gtcgggaggc ggtggggcgc gctcctctct gcctaccctc ctcacagcct 2640

catgaacccc aggtctgtgg gagcctcctc catggggcca cacggtcctt ggcctcaccc 2700

cctgttttga agatggggca ctgaggccgg agaggggtaa ggcctcgctc gagtccaggt 2760

ccccagaggc tgagcccaga gtaatcttga accaccccca ttcagggtct ggcctggagg 2820

agcctgaccc acagaggaga caccctggga gatattcatt gaggggtaat ctggtccccc 2880

gcaaatccag gggtgattcc cactgcccca taggcacagc cacgtggaag aaggcaggca 2940

atgttggggc tcctcacttc ctagaggcct cacaactcaa atgcccccca ctgcagctgg 3000

gggtggggtg gtggtatggg atggggacca agccttcctt gaaggataga gcccagccca 3060

acaccccgcc ccgtggcagc agcatcacgt gttccagcga ggaaggagag caccagactc 3120

agtcatgatc actgttgcct tgaacttcca agaacagccc cagggcaagg gtcaaaacag 3180

gggaaagggg gtgatgagag atccttcttc cggatgttcc tccaggaacc agggggctgg 3240

ctggtcttgg ctgggttcgg gtaggagacc catgatgaat aaacttggga atcactgggg 3300

tggctgtaag ggaatttagg ggagctccga aggggccctt aggctcgagg agatgctcct 3360

ctcttttccc gaattcccag ggacccagga gagtgtccct tcttcctctt cctgtgtgtc 3420

catccacccc cgccccccgc cctggcagag ctggtggaac tcagtgctct agcccctacc 3480

ctggggttgc gactctggct caggacacca ccacgctccc tgggggtgtg agtgagggcc 3540

tgtgcgctcc atcccgagtg ctgcctgttt cagctaaagc ctcaaagcaa gagaaacccc 3600

ctctctaagc ggcccctcag ccatcgggtg ggtcgtttgg tttctgggta ggcctcaggg 3660

gctggccacc tgcagggccc agcccaaccc agggatgcag atgtcccagc cacatccctg 3720

tcccagtttc ctgctcccca aggcatccac cctgctgttg gtgcgagggc tgatagaggg 3780

cacgccaagt cactcccctg cccttccctc cttccagccc tgtgctccgg ccaggtcttc 3840

acccagaggt ctggggagct cagcagccct gaatacccac ggccgtatcc caaactctcc 3900

agttgcactt acagcatcag cctggaggag gggttcagtg tcattctgga ctttgtggag 3960

tccttcgatg tggagacaca ccctgaaacc ctgtgtccct acgactttct caaggtctgg 4020

ctcctgggcc cctcatcttg tcccagatcc tcccccttca gcccagctgc accccctact 4080

tcctgcagca tggcccccac cacgttcccg tcaccctcgg tgaccccacc tcttcaggtg 4140

ctctatggag gtcaaggctg gggcttcgag tacaagtgtg ggaggcagag tggggagggg 4200

caccccaatc catggcctgg gttggcctca ttggctgtcc ctgaaatgct gaggaggtgg 4260

gttacttccc tccgcccagg ccagacccag gcagctgctc cccagctttc atgagcttct 4320

ttctcagatt caaacagaca gagaagaaca tggcccattc tgtgggaaga cattgcccca 4380

caggattgaa acaaaaagca acacggtgac catcaccttt gtcacagatg aatcaggaga 4440

ccacacaggc tggaagatcc actacacgag cacagtgagc aagtgggctc agatccttgg 4500

tggaagcgca gagctgcctc tctctggagt gcaaggagct gtagagtgta gggctcttct 4560

gggcaggact aggaagggac accaggttta gtggtgctga ggtctgaggc agcagcttct 4620

aaggggaagc acccgtgccc tcctcagcag cacccagcat cttcaccact cattcttcaa 4680

ccacccattc acccatcact catcttttac ccacccaccc tttgccactc atccttctgt 4740

ccctcatcct tccaaccatt catcaatcac ccacccatcc atcctttgcc acacaaccat 4800

ccacccattc ttctacctac ccatcctatc catccatcct tctatcagca tccttctacc 4860

acccatcctt cgttcggtca tccatcatca tccatccatc 4900

<210> 8

<211> 136

<212> PRT

<213> Chile person

<400> 8

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr

1 5 10 15

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

20 25 30

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser

100 105 110

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala

130 135

<210> 9

<211> 181

<212> PRT

<213> Chile person

<400> 9

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr

1 5 10 15

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

20 25 30

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser

100 105 110

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val

130 135 140

Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu

145 150 155 160

Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn

165 170 175

Lys Arg Thr Cys Ser

180

<210> 10

<211> 293

<212> PRT

<213> Chile person

<400> 10

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr

1 5 10 15

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

20 25 30

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser

100 105 110

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val

130 135 140

Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu

145 150 155 160

Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn

165 170 175

Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg

180 185 190

Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu

195 200 205

Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile

210 215 220

Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu

225 230 235 240

Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly

245 250 255

Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn

260 265 270

Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly

275 280 285

Trp Lys Ile His Tyr

290

<210> 11

<211> 41

<212> PRT

<213> Chile person

<400> 11

Glu Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys

1 5 10 15

Asp His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg

20 25 30

Ala Gly Tyr Val Leu His Arg Asn Lys

35 40

<210> 12

<211> 242

<212> PRT

<213> Chile person

<400> 12

Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val

1 5 10 15

Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn

20 25 30

Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala

35 40 45

Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His

50 55 60

Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr

65 70 75 80

His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn

85 90 95

Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys

100 105 110

Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly

115 120 125

Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val

130 135 140

Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys

145 150 155 160

Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly

165 170 175

Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala

180 185 190

Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile

195 200 205

Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val

210 215 220

Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser

225 230 235 240

Asp Phe

<210> 13

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 13

Gly Lys Asp Ser Cys Arg Gly Asp Ala Gly Gly Ala Leu Val Phe Leu

1 5 10 15

<210> 14

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 14

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu

1 5 10 15

<210> 15

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 15

Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe Asp

1 5 10 15

Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser

20 25 30

Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln

35 40

<210> 16

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 16

Thr Phe Arg Ser Asp Tyr Ser Asn

1 5

<210> 17

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 17

Phe Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr

1 5 10 15

Ser Asn Glu Lys Pro Phe Thr Gly Phe

20 25

<210> 18

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 18

Ile Asp Glu Cys Gln Val Ala Pro Gly

1 5

<210> 19

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 19

Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys

1 5 10 15

Arg Gly Asp Ser Gly Gly Ala Leu Val

20 25

<210> 20

<211> 960

<212> DNA

<213> Chile person

<220>

<221> CDS

<222> (51)..(797)

<400> 20

attaactgag attaaccttc cctgagtttt ctcacaccaa ggtgaggacc atg tcc 56

Met Ser

1

ctg ttt cca tca ctc cct ctc ctt ctc ctg agt atg gtg gca gcg tct 104

Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala Ala Ser

5 10 15

tac tca gaa act gtg acc tgt gag gat gcc caa aag acc tgc cct gca 152

Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala

20 25 30

gtg att gcc tgt agc tct cca ggc atc aac ggc ttc cca ggc aaa gat 200

Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp

35 40 45 50

ggg cgt gat ggc acc aag gga gaa aag ggg gaa cca ggc caa ggg ctc 248

Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln Gly Leu

55 60 65

aga ggc tta cag ggc ccc cct gga aag ttg ggg cct cca gga aat cca 296

Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn Pro

70 75 80

ggg cct tct ggg tca cca gga cca aag ggc caa aaa gga gac cct gga 344

Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp Pro Gly

85 90 95

aaa agt ccg gat ggt gat agt agc ctg gct gcc tca gaa aga aaa gct 392

Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala

100 105 110

ctg caa aca gaa atg gca cgt atc aaa aag tgg ctc acc ttc tct ctg 440

Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu

115 120 125 130

ggc aaa caa gtt ggg aac aag ttc ttc ctg acc aat ggt gaa ata atg 488

Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met

135 140 145

acc ttt gaa aaa gtg aag gcc ttg tgt gtc aag ttc cag gcc tct gtg 536

Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val

150 155 160

gcc acc ccc agg aat gct gca gag aat gga gcc att cag aat ctc atc 584

Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile

165 170 175

aag gag gaa gcc ttc ctg ggc atc act gat gag aag aca gaa ggg cag 632

Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln

180 185 190

ttt gtg gat ctg aca gga aat aga ctg acc tac aca aac tgg aac gag 680

Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu

195 200 205 210

ggt gaa ccc aac aat gct ggt tct gat gaa gat tgt gta ttg cta ctg 728

Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu

215 220 225

aaa aat ggc cag tgg aat gac gtc ccc tgc tcc acc tcc cat ctg gcc 776

Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala

230 235 240

gtc tgt gag ttc cct atc tga agggtcatat cactcaggcc ctccttgtct 827

Val Cys Glu Phe Pro Ile

245

ttttactgca acccacaggc ccacagtatg cttgaaaaga taaattatat caatttcctc 887

atatccagta ttgttccttt tgtgggcaat cactaaaaat gatcactaac agcaccaaca 947

aagcaataat agt 960

<210> 21

<211> 248

<212> PRT

<213> Chile person

<400> 21

Met Ser Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala

1 5 10 15

Ala Ser Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys

20 25 30

Pro Ala Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly

35 40 45

Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln

50 55 60

Gly Leu Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly

65 70 75 80

Asn Pro Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp

85 90 95

Pro Gly Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg

100 105 110

Lys Ala Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe

115 120 125

Ser Leu Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu

130 135 140

Ile Met Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala

145 150 155 160

Ser Val Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn

165 170 175

Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu

180 185 190

Gly Gln Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp

195 200 205

Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu

210 215 220

Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His

225 230 235 240

Leu Ala Val Cys Glu Phe Pro Ile

245

<210> 22

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> wherein x at position 1 represents hydroxyproline

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> wherein X at position 4 represents a hydrophobic residue

<400> 22

Xaa Gly Lys Xaa Gly Pro

1 5

<210> 23

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> wherein X represents hydroxyproline

<400> 23

Xaa Gly Lys Leu Gly

1 5

<210> 24

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (9)..(15)

<223> wherein X at positions 9 and 15 represents hydroxyproline

<400> 24

Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa Gly

1 5 10 15

<210> 25

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (3)..(27)

<223> wherein X at positions 3, 6, 15, 21, 24, 27 represents hydroxyproline

<400> 25

Gly Pro Xaa Gly Pro Xaa Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly

1 5 10 15

Lys Leu Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa

20 25

<210> 26

<211> 53

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> misc_feature

<222> (26)..(26)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (41)..(41)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (50)..(50)

<223> Xaa can be any naturally occurring amino acid

<400> 26

Gly Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly

1 5 10 15

Gln Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa

20 25 30

Gly Asn Xaa Gly Pro Ser Gly Ser Xaa Gly Pro Lys Gly Gln Lys Gly

35 40 45

Asp Xaa Gly Lys Ser

50

<210> 27

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (3)..(33)

<223> wherein X at positions 3, 6, 12, 18, 21, 30, 33 represents hydroxyproline

<400> 27

Gly Ala Xaa Gly Ser Xaa Gly Glu Lys Gly Ala Xaa Gly Pro Gln Gly

1 5 10 15

Pro Xaa Gly Pro Xaa Gly Lys Met Gly Pro Lys Gly Glu Xaa Gly Asp

20 25 30

Xaa

<210> 28

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (3)..(45)

<223> wherein X at positions 3, 6, 9, 27, 30, 36, 42, 45 represents hydroxyproline

<400> 28

Gly Cys Xaa Gly Leu Xaa Gly Ala Xaa Gly Asp Lys Gly Glu Ala Gly

1 5 10 15

Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Xaa Gly Pro Xaa Gly Lys

20 25 30

Ala Gly Pro Xaa Gly Pro Asn Gly Ala Xaa Gly Glu Xaa

35 40 45

<210> 29

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 29

Leu Gln Arg Ala Leu Glu Ile Leu Pro Asn Arg Val Thr Ile Lys Ala

1 5 10 15

Asn Arg Pro Phe Leu Val Phe Ile

20

<210> 30

<211> 559

<212> DNA

<213> Chile person

<400> 30

atgaggctgc tgaccctcct gggccttctg tgtggctcgg tggccacccc cttgggcccg 60

aagtggcctg aacctgtgtt cgggcgcctg gcatcccccg gctttccagg ggagtatgcc 120

aatgaccagg agcggcgctg gaccctgact gcaccccccg gctaccgcct gcgcctctac 180

ttcacccact tcgacctgga gctctcccac ctctgcgagt acgacttcgt caagctgagc 240

tcgggggcca aggtgctggc cacgctgtgc gggcaggaga gcacagacac ggagcgggcc 300

cctggcaagg acactttcta ctcgctgggc tccagcctgg acattacctt ccgctccgac 360

tactccaacg agaagccgtt cacggggttc gaggccttct atgcagccga ggacattgac 420

gagtgccagg tggccccggg agaggcgccc acctgcgacc accactgcca caaccacctg 480

ggcggtttct actgctcctg ccgcgcaggc tacgtcctgc accgtaacaa gcgcacctgc 540

tcagccctgt gctccggcc 559

<210> 31

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 31

cgggcacacc atgaggctgc tgaccctcct gggc 34

<210> 32

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 32

gacattacct tccgctccga ctccaacgag aag 33

<210> 33

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 33

agcagccctg aatacccacg gccgtatccc aaa 33

<210> 34

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 34

cgggatccat gaggctgctg accctc 26

<210> 35

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 35

ggaattccta ggctgcata 19

<210> 36

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 36

ggaattccta cagggcgct 19

<210> 37

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 37

ggaattccta gtagtggat 19

<210> 38

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 38

tgcggccgct gtaggtgctg tcttt 25

<210> 39

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 39

ggaattcact cgttattctc gga 23

<210> 40

<211> 17

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 40

tccgagaata acgagtg 17

<210> 41

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 41

cattgaaagc tttggggtag aagttgttc 29

<210> 42

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 42

cgcggccgca gctgctcaga gtgtaga 27

<210> 43

<211> 28

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 43

cggtaagctt cactggctca gggaaata 28

<210> 44

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 44

aagaagcttg ccgccaccat ggattggctg tggaact 37

<210> 45

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 45

cgggatcctc aaactttctt gtccaccttg g 31

<210> 46

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 46

aagaaagctt gccgccacca tgttctcact agctct 36

<210> 47

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 47

cgggatcctt ctccctctaa cactct 26

<210> 48

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 48

Glu Pro Lys Ser Cys Asp Lys Thr His

1 5

<210> 49

<211> 4960

<212> DNA

<213> Chile person

<400> 49

ccggacgtgg tggcgcatgc ctgtaatccc agctactcgg gaggctgagg caggagaatt 60

gctcgaaccc cggaggcaga ggtttggtgg ctcacacctg taatcccagc actttgcgag 120

gctgaggcag gtgcatcgct ttggctcagg agttcaagac cagcctgggc aacacaggga 180

gacccccatc tctacaaaaa acaaaaacaa atataaaggg gataaaaaaa aaaaaaagac 240

aagacatgaa tccatgagga cagagtgtgg aagaggaagc agcagcctca aagttctgga 300

agctggaaga acagataaac aggtgtgaaa taactgcctg gaaagcaact tctttttttt 360

tttttttttt tttgaggtgg agtctcactc tgtcgtccag gctggagtgc agtggtgcga 420

tctcggatca ctgcaacctc cgcctcccag gctcaagcaa ttctcctgcc tcagcctccc 480

gagtagctgg gattataagt gcgcgctgcc acacctggat gatttttgta tttttagtag 540

agatgggatt tcaccatgtt ggtcaggctg gtctcaaact cccaacctcg tgatccaccc 600

accttggcct cccaaagtgc tgggattaca ggtataagcc accgagccca gccaaaagcg 660

acttctaagc ctgcaaggga atcgggaatt ggtggcacca ggtccttctg acagggttta 720

agaaattagc cagcctgagg ctgggcacgg tggctcacac ctgtaatccc agcactttgg 780

gaggctaagg caggtggatc acctgagggc aggagttcaa gaccagcctg accaacatgg 840

agaaacccca tccctaccaa aaataaaaaa ttagccaggt gtggtggtgc tcgcctgtaa 900

tcccagctac ttgggaggct gaggtgggag gattgcttga acacaggaag tagaggctgc 960

agtgagctat gattgcagca ctgcactgaa gccggggcaa cagaacaaga tccaaaaaaa 1020

agggaggggt gaggggcaga gccaggattt gtttccaggc tgttgttacc taggtccgac 1080

tcctggctcc cagagcagcc tgtcctgcct gcctggaact ctgagcaggc tggagtcatg 1140

gagtcgattc ccagaatccc agagtcaggg aggctggggg caggggcagg tcactggaca 1200

aacagatcaa aggtgagacc agcgtagggc tgcagaccag gccaggccag ctggacgggc 1260

acaccatgag gtaggtgggc gcccacagcc tccctgcagg gtgtggggtg ggagcacagg 1320

cctgggccct caccgcccct gccctgccca taggctgctg accctcctgg gccttctgtg 1380

tggctcggtg gccaccccct tgggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc 1440

atcccccggc tttccagggg agtatgccaa tgaccaggag cggcgctgga ccctgactgc 1500

accccccggc taccgcctgc gcctctactt cacccacttc gacctggagc tctcccacct 1560

ctgcgagtac gacttcgtca aggtgccgtc aggacgggag ggctggggtt tctcagggtc 1620

ggggggtccc caaggagtag ccagggttca gggacacctg ggagcagggg ccaggcttgg 1680

ccaggaggga gatcaggcct gggtcttgcc ttcactccct gtgacacctg accccacagc 1740

tgagctcggg ggccaaggtg ctggccacgc tgtgcgggca ggagagcaca gacacggagc 1800

gggcccctgg caaggacact ttctactcgc tgggctccag cctggacatt accttccgct 1860

ccgactactc caacgagaag ccgttcacgg ggttcgaggc cttctatgca gccgagggtg 1920

agccaagagg ggtcctgcaa catctcagtc tgcgcagctg gctgtggggg taactctgtc 1980

ttaggccagg cagccctgcc ttcagtttcc ccacctttcc cagggcaggg gagaggcctc 2040

tggcctgaca tcatccacaa tgcaaagacc aaaacagccg tgacctccat tcacatgggc 2100

tgagtgccaa ctctgagcca gggatctgag gacagcatcg cctcaagtga cgcagggact 2160

ggccgggcgc agcagctcac gcctgtaatt ccagcacttt gggaggccga ggctggctga 2220

tcatttgagg tcaggagttc aaggccagcc agggcaacac ggtgaaactc tatctccact 2280

aaaactacaa aaattagctg ggcgtggtgg tgcgcacctg gaatcccagc tactagggag 2340

gctgaggcag gagaattgct tgaacctgcg aggtggaggc tgcagtgaac agagattgca 2400

ccactacact ccagcctggg cgacagagct agactccgtc tcaaaaaaca aaaaacaaaa 2460

acgacgcagg ggccgagggc cccatttaca gctgacaaag tggggccctg ccagcgggag 2520

cgctgccagg atgtttgatt tcagatccca gtccctgcag agaccaactg tgtgacctct 2580

ggcaagtggc tcaatttctc tgctccttag gaagctgctg caagggttca gcgctgtagc 2640

cccgccccct gggtttgatt gactcccctc attagctggg tgacctcggg ccggacactg 2700

aaactcccac tggtttaaca gaggtgatgt ttgcatcttt ctcccagcgc tgctgggagc 2760

ttgcagcgac cctaggcctg taaggtgatt ggcccggcac cagtcccgca ccctagacag 2820

gacgaggcct cctctgaggt ccactctgag gtcatggatc tcctgggagg agtccaggct 2880

ggatcccgcc tctttccctc ctgacggcct gcctggccct gcctctcccc cagacattga 2940

cgagtgccag gtggccccgg gagaggcgcc cacctgcgac caccactgcc acaaccacct 3000

gggcggtttc tactgctcct gccgcgcagg ctacgtcctg caccgtaaca agcgcacctg 3060

ctcagccctg tgctccggcc aggtcttcac ccagaggtct ggggagctca gcagccctga 3120

atacccacgg ccgtatccca aactctccag ttgcacttac agcatcagcc tggaggaggg 3180

gttcagtgtc attctggact ttgtggagtc cttcgatgtg gagacacacc ctgaaaccct 3240

gtgtccctac gactttctca agattcaaac agacagagaa gaacatggcc cattctgtgg 3300

gaagacattg ccccacagga ttgaaacaaa aagcaacacg gtgaccatca cctttgtcac 3360

agatgaatca ggagaccaca caggctggaa gatccactac acgagcacag cgcacgcttg 3420

cccttatccg atggcgccac ctaatggcca cgtttcacct gtgcaagcca aatacatcct 3480

gaaagacagc ttctccatct tttgcgagac tggctatgag cttctgcaag gtcacttgcc 3540

cctgaaatcc tttactgcag tttgtcagaa agatggatct tgggaccggc caatgcccgc 3600

gtgcagcatt gttgactgtg gccctcctga tgatctaccc agtggccgag tggagtacat 3660

cacaggtcct ggagtgacca cctacaaagc tgtgattcag tacagctgtg aagagacctt 3720

ctacacaatg aaagtgaatg atggtaaata tgtgtgtgag gctgatggat tctggacgag 3780

ctccaaagga gaaaaatcac tcccagtctg tgagcctgtt tgtggactat cagcccgcac 3840

aacaggaggg cgtatatatg gagggcaaaa ggcaaaacct ggtgattttc cttggcaagt 3900

cctgatatta ggtggaacca cagcagcagg tgcactttta tatgacaact gggtcctaac 3960

agctgctcat gccgtctatg agcaaaaaca tgatgcatcc gccctggaca ttcgaatggg 4020

caccctgaaa agactatcac ctcattatac acaagcctgg tctgaagctg tttttataca 4080

tgaaggttat actcatgatg ctggctttga caatgacata gcactgatta aattgaataa 4140

caaagttgta atcaatagca acatcacgcc tatttgtctg ccaagaaaag aagctgaatc 4200

ctttatgagg acagatgaca ttggaactgc atctggatgg ggattaaccc aaaggggttt 4260

tcttgctaga aatctaatgt atgtcgacat accgattgtt gaccatcaaa aatgtactgc 4320

tgcatatgaa aagccaccct atccaagggg aagtgtaact gctaacatgc tttgtgctgg 4380

cttagaaagt gggggcaagg acagctgcag aggtgacagc ggaggggcac tggtgtttct 4440

agatagtgaa acagagaggt ggtttgtggg aggaatagtg tcctggggtt ccatgaattg 4500

tggggaagca ggtcagtatg gagtctacac aaaagttatt aactatattc cctggatcga 4560

gaacataatt agtgattttt aacttgcgtg tctgcagtca aggattcttc atttttagaa 4620

atgcctgtga agaccttggc agcgacgtgg ctcgagaagc attcatcatt actgtggaca 4680

tggcagttgt tgctccaccc aaaaaaacag actccaggtg aggctgctgt catttctcca 4740

cttgccagtt taattccagc cttacccatt gactcaaggg gacataaacc acgagagtga 4800

cagtcatctt tgcccaccca gtgtaatgtc actgctcaaa ttacatttca ttaccttaaa 4860

aagccagtct cttttcatac tggctgttgg catttctgta aactgcctgt ccatgctctt 4920

tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 4960

<210> 50

<211> 2090

<212> DNA

<213> mouse

<220>

<221> CDS

<222> (33)..(2090)

<400> 50

ggcgctggac tgcagagcta tggtggcaca cc atg agg cta ctc atc ttc ctg 53

Met Arg Leu Leu Ile Phe Leu

1 5

ggt ctg ctg tgg agt ttg gtg gcc aca ctt ctg ggt tca aag tgg cct 101

Gly Leu Leu Trp Ser Leu Val Ala Thr Leu Leu Gly Ser Lys Trp Pro

10 15 20

gaa cct gta ttc ggg cgc ctg gtg tcc cct ggc ttc cca gag aag tat 149

Glu Pro Val Phe Gly Arg Leu Val Ser Pro Gly Phe Pro Glu Lys Tyr

25 30 35

gct gac cat caa gat cga tcc tgg aca ctg act gca ccc cct ggc tac 197

Ala Asp His Gln Asp Arg Ser Trp Thr Leu Thr Ala Pro Pro Gly Tyr

40 45 50 55

cgc ctg cgc ctc tac ttc acc cac ttt gac ctg gaa ctc tct tac cgc 245

Arg Leu Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser Tyr Arg

60 65 70

tgc gag tat gac ttt gtc aag ttg agc tca ggg acc aag gtg ctg gcc 293

Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser Gly Thr Lys Val Leu Ala

75 80 85

aca ctg tgt ggg cag gag agt aca gac act gag cag gca cct ggc aat 341

Thr Leu Cys Gly Gln Glu Ser Thr Asp Thr Glu Gln Ala Pro Gly Asn

90 95 100

gac acc ttc tac tca ctg ggt ccc agc cta aag gtc acc ttc cac tcc 389

Asp Thr Phe Tyr Ser Leu Gly Pro Ser Leu Lys Val Thr Phe His Ser

105 110 115

gac tac tcc aat gag aag ccg ttc aca ggg ttt gag gcc ttc tat gca 437

Asp Tyr Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala

120 125 130 135

gcg gag gat gtg gat gaa tgc aga gtg tct ctg gga gac tca gtc cct 485

Ala Glu Asp Val Asp Glu Cys Arg Val Ser Leu Gly Asp Ser Val Pro

140 145 150

tgt gac cat tat tgc cac aac tac ttg ggc ggc tac tat tgc tcc tgc 533

Cys Asp His Tyr Cys His Asn Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys

155 160 165

aga gcg ggc tac att ctc cac cag aac aag cac acg tgc tca gcc ctt 581

Arg Ala Gly Tyr Ile Leu His Gln Asn Lys His Thr Cys Ser Ala Leu

170 175 180

tgt tca ggc cag gtg ttc aca gga aga tct ggg tat ctc agt agc cct 629

Cys Ser Gly Gln Val Phe Thr Gly Arg Ser Gly Tyr Leu Ser Ser Pro

185 190 195

gag tac ccg cag cca tac ccc aag ctc tcc agc tgc acc tac agc atc 677

Glu Tyr Pro Gln Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr Ser Ile

200 205 210 215

cgc ctg gag gac ggc ttc agt gtc atc ctg gac ttc gtg gag tcc ttc 725

Arg Leu Glu Asp Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe

220 225 230

gat gtg gag acg cac cct gaa gcc cag tgc ccc tat gac tcc ctc aag 773

Asp Val Glu Thr His Pro Glu Ala Gln Cys Pro Tyr Asp Ser Leu Lys

235 240 245

att caa aca gac aag ggg gaa cac ggc cca ttt tgt ggg aag acg ctg 821

Ile Gln Thr Asp Lys Gly Glu His Gly Pro Phe Cys Gly Lys Thr Leu

250 255 260

cct ccc agg att gaa act gac agc cac aag gtg acc atc acc ttt gcc 869

Pro Pro Arg Ile Glu Thr Asp Ser His Lys Val Thr Ile Thr Phe Ala

265 270 275

act gac gag tcg ggg aac cac aca ggc tgg aag ata cac tac aca agc 917

Thr Asp Glu Ser Gly Asn His Thr Gly Trp Lys Ile His Tyr Thr Ser

280 285 290 295

aca gca cgg ccc tgc cct gat cca acg gcg cca cct aat ggc agc att 965

Thr Ala Arg Pro Cys Pro Asp Pro Thr Ala Pro Pro Asn Gly Ser Ile

300 305 310

tca cct gtg caa gcc acg tat gtc ctg aag gac agg ttt tct gtc ttc 1013

Ser Pro Val Gln Ala Thr Tyr Val Leu Lys Asp Arg Phe Ser Val Phe

315 320 325

tgc aag aca ggc ttc gag ctt ctg caa ggt tct gtc ccc ctg aaa tca 1061

Cys Lys Thr Gly Phe Glu Leu Leu Gln Gly Ser Val Pro Leu Lys Ser

330 335 340

ttc act gct gtc tgt cag aaa gat gga tct tgg gac cgg ccg atg cca 1109

Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro

345 350 355

gag tgc agc att att gat tgt ggc cct ccc gat gac cta ccc aat ggc 1157

Glu Cys Ser Ile Ile Asp Cys Gly Pro Pro Asp Asp Leu Pro Asn Gly

360 365 370 375

cat gtg gac tat atc aca ggc cct caa gtg act acc tac aaa gct gtg 1205

His Val Asp Tyr Ile Thr Gly Pro Gln Val Thr Thr Tyr Lys Ala Val

380 385 390

att cag tac agc tgt gaa gag act ttc tac aca atg agc agc aat ggt 1253

Ile Gln Tyr Ser Cys Glu Glu Thr Phe Tyr Thr Met Ser Ser Asn Gly

395 400 405

aaa tat gtg tgt gag gct gat gga ttc tgg acg agc tcc aaa gga gaa 1301

Lys Tyr Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu

410 415 420

aaa ctc ccc ccg gtt tgt gag cct gtt tgt ggg ctg tcc aca cac act 1349

Lys Leu Pro Pro Val Cys Glu Pro Val Cys Gly Leu Ser Thr His Thr

425 430 435

ata gga gga cgc ata gtt gga ggg cag cct gca aag cct ggt gac ttt 1397

Ile Gly Gly Arg Ile Val Gly Gly Gln Pro Ala Lys Pro Gly Asp Phe

440 445 450 455

cct tgg caa gtc ttg ttg ctg ggt caa act aca gca gca gca ggt gca 1445

Pro Trp Gln Val Leu Leu Leu Gly Gln Thr Thr Ala Ala Ala Gly Ala

460 465 470

ctt ata cat gac aat tgg gtc cta aca gcc gct cat gct gta tat gag 1493

Leu Ile His Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu

475 480 485

aaa aga atg gca gcg tcc tcc ctg aac atc cga atg ggc atc ctc aaa 1541

Lys Arg Met Ala Ala Ser Ser Leu Asn Ile Arg Met Gly Ile Leu Lys

490 495 500

agg ctc tca cct cat tac act caa gcc tgg ccc gag gaa atc ttt ata 1589

Arg Leu Ser Pro His Tyr Thr Gln Ala Trp Pro Glu Glu Ile Phe Ile

505 510 515

cat gaa ggc tac act cac ggt gct ggt ttt gac aat gat ata gca ttg 1637

His Glu Gly Tyr Thr His Gly Ala Gly Phe Asp Asn Asp Ile Ala Leu

520 525 530 535

att aaa ctc aag aac aaa gtc aca atc aac gga agc atc atg cct gtt 1685

Ile Lys Leu Lys Asn Lys Val Thr Ile Asn Gly Ser Ile Met Pro Val

540 545 550

tgc cta ccg cga aaa gaa gct gca tcc tta atg aga aca gac ttc act 1733

Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu Met Arg Thr Asp Phe Thr

555 560 565

gga act gtg gct ggc tgg ggg tta acc cag aag ggg ctt ctt gct aga 1781

Gly Thr Val Ala Gly Trp Gly Leu Thr Gln Lys Gly Leu Leu Ala Arg

570 575 580

aac cta atg ttt gtg gac ata cca att gct gac cac caa aaa tgt acc 1829

Asn Leu Met Phe Val Asp Ile Pro Ile Ala Asp His Gln Lys Cys Thr

585 590 595

acc gtg tat gaa aag ctc tat cca gga gta aga gta agc gct aac atg 1877

Thr Val Tyr Glu Lys Leu Tyr Pro Gly Val Arg Val Ser Ala Asn Met

600 605 610 615

ctc tgt gct ggc tta gag act ggt ggc aag gac agc tgc aga ggt gac 1925

Leu Cys Ala Gly Leu Glu Thr Gly Gly Lys Asp Ser Cys Arg Gly Asp

620 625 630

agt ggg ggg gca tta gtg ttt cta gat aat gag aca cag cga tgg ttt 1973

Ser Gly Gly Ala Leu Val Phe Leu Asp Asn Glu Thr Gln Arg Trp Phe

635 640 645

gtg gga gga ata gtt tcc tgg ggt tcc att aat tgt ggg gcg gca ggc 2021

Val Gly Gly Ile Val Ser Trp Gly Ser Ile Asn Cys Gly Ala Ala Gly

650 655 660

cag tat ggg gtc tac aca aaa gtc atc aac tat att ccc tgg aat gag 2069

Gln Tyr Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Asn Glu

665 670 675

aac ata ata agt aat ttc taa 2090

Asn Ile Ile Ser Asn Phe

680 685

<210> 51

<211> 685

<212> PRT

<213> mouse

<400> 51

Met Arg Leu Leu Ile Phe Leu Gly Leu Leu Trp Ser Leu Val Ala Thr

1 5 10 15

Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser

20 25 30

Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser

100 105 110

Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Val

130 135 140

Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu

145 150 155 160

Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln Asn

165 170 175

Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg

180 185 190

Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu

195 200 205

Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val Ile

210 215 220

Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala Gln

225 230 235 240

Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His Gly

245 250 255

Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser His

260 265 270

Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr Gly

275 280 285

Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro Thr

290 295 300

Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val Leu

305 310 315 320

Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln

325 330 335

Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly

340 345 350

Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Ile Asp Cys Gly Pro

355 360 365

Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Gln

370 375 380

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe

385 390 395 400

Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe

405 410 415

Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro Val

420 425 430

Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly Gln

435 440 445

Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Gln

450 455 460

Thr Thr Ala Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu Thr

465 470 475 480

Ala Ala His Ala Val Tyr Glu Lys Arg Met Ala Ala Ser Ser Leu Asn

485 490 495

Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala

500 505 510

Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly

515 520 525

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile

530 535 540

Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala Ser

545 550 555 560

Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu Thr

565 570 575

Gln Lys Gly Leu Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile

580 585 590

Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro Gly

595 600 605

Val Arg Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly Gly

610 615 620

Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp

625 630 635 640

Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser

645 650 655

Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val Ile

660 665 670

Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe

675 680 685

<210> 52

<211> 670

<212> PRT

<213> mouse

<400> 52

Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val

1 5 10 15

Ser Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp

20 25 30

Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Ser Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr

65 70 75 80

Asp Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro

85 90 95

Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg

115 120 125

Val Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr

130 135 140

Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln

145 150 155 160

Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly

165 170 175

Arg Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys

180 185 190

Leu Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val

195 200 205

Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala

210 215 220

Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His

225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser

245 250 255

His Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr

260 265 270

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro

275 280 285

Thr Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val

290 295 300

Leu Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu

305 310 315 320

Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp

325 330 335

Gly Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Ile Asp Cys Gly

340 345 350

Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro

355 360 365

Gln Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr

370 375 380

Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly

385 390 395 400

Phe Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro

405 410 415

Val Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly

420 425 430

Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly

435 440 445

Gln Thr Thr Ala Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu

450 455 460

Thr Ala Ala His Ala Val Tyr Glu Lys Arg Met Ala Ala Ser Ser Leu

465 470 475 480

Asn Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln

485 490 495

Ala Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala

500 505 510

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr

515 520 525

Ile Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala

530 535 540

Ser Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu

545 550 555 560

Thr Gln Lys Gly Leu Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro

565 570 575

Ile Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro

580 585 590

Gly Val Arg Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly

595 600 605

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu

610 615 620

Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly

625 630 635 640

Ser Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val

645 650 655

Ile Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe

660 665 670

<210> 53

<211> 2091

<212> DNA

<213> rat

<220>

<221> CDS

<222> (10)..(2067)

<400> 53

tggcacaca atg agg cta ctg atc gtc ctg ggt ctg ctt tgg agt ttg gtg 51

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val

1 5 10

gcc aca ctt ttg ggc tcc aag tgg cct gag cct gta ttc ggg cgc ctg 99

Ala Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu

15 20 25 30

gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc 147

Val Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser

35 40 45

tgg acg ctg act gca ccc cct ggc ttc cgc ctg cgc ctc tac ttc acc 195

Trp Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr

50 55 60

cac ttc aac ctg gaa ctc tct tac cgc tgc gag tat gac ttt gtc aag 243

His Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys

65 70 75

ttg acc tca ggg acc aag gtg cta gcc acg ctg tgt ggg cag gag agt 291

Leu Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser

80 85 90

aca gat act gag cgg gca cct ggc aat gac acc ttc tac tca ctg ggt 339

Thr Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly

95 100 105 110

ccc agc cta aag gtc acc ttc cac tcc gac tac tcc aat gag aag cca 387

Pro Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro

115 120 125

ttc aca gga ttt gag gcc ttc tat gca gcg gag gat gtg gat gaa tgc 435

Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys

130 135 140

aga aca tcc ctg gga gac tca gtc cct tgt gac cat tat tgc cac aac 483

Arg Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn

145 150 155

tac ctg ggc ggc tac tac tgc tcc tgc cga gtg ggc tac att ctg cac 531

Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His

160 165 170

cag aac aag cat acc tgc tca gcc ctt tgt tca ggc cag gtg ttc act 579

Gln Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr

175 180 185 190

ggg agg tct ggc ttt ctc agt agc cct gag tac cca cag cca tac ccc 627

Gly Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro

195 200 205

aaa ctc tcc agc tgc gcc tac aac atc cgc ctg gag gaa ggc ttc agt 675

Lys Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser

210 215 220

atc acc ctg gac ttc gtg gag tcc ttt gat gtg gag atg cac cct gaa 723

Ile Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu

225 230 235

gcc cag tgc ccc tac gac tcc ctc aag att caa aca gac aag agg gaa 771

Ala Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu

240 245 250

tac ggc ccg ttt tgt ggg aag acg ctg ccc ccc agg att gaa act gac 819

Tyr Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp

255 260 265 270

agc aac aag gtg acc att acc ttt acc acc gac gag tca ggg aac cac 867

Ser Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His

275 280 285

aca ggc tgg aag ata cac tac aca agc aca gca cag ccc tgc cct gat 915

Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp

290 295 300

cca acg gcg cca cct aat ggt cac att tca cct gtg caa gcc acg tat 963

Pro Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr

305 310 315

gtc ctg aag gac agc ttt tct gtc ttc tgc aag act ggc ttc gag ctt 1011

Val Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu

320 325 330

ctg caa ggt tct gtc ccc ctg aag tca ttc act gct gtc tgt cag aaa 1059

Leu Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys

335 340 345 350

gat gga tct tgg gac cgg ccg ata cca gag tgc agc att att gac tgt 1107

Asp Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys

355 360 365

ggc cct ccc gat gac cta ccc aat ggc cac gtg gac tat atc aca ggc 1155

Gly Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly

370 375 380

cct gaa gtg acc acc tac aaa gct gtg att cag tac agc tgt gaa gag 1203

Pro Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu

385 390 395

act ttc tac aca atg agc agc aat ggt aaa tat gtg tgt gag gct gat 1251

Thr Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp

400 405 410

gga ttc tgg acg agc tcc aaa gga gaa aaa tcc ctc ccg gtt tgc aag 1299

Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys

415 420 425 430

cct gtc tgt gga ctg tcc aca cac act tca gga ggc cgt ata att gga 1347

Pro Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly

435 440 445

gga cag cct gca aag cct ggt gac ttt cct tgg caa gtc ttg tta ctg 1395

Gly Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu

450 455 460

ggt gaa act aca gca gca ggt gct ctt ata cat gac gac tgg gtc cta 1443

Gly Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu

465 470 475

aca gcg gct cat gct gta tat ggg aaa aca gag gcg atg tcc tcc ctg 1491

Thr Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu

480 485 490

gac atc cgc atg ggc atc ctc aaa agg ctc tcc ctc att tac act caa 1539

Asp Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln

495 500 505 510

gcc tgg cca gag gct gtc ttt atc cat gaa ggc tac act cac gga gct 1587

Ala Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala

515 520 525

ggt ttt gac aat gat ata gca ctg att aaa ctc aag aac aaa gtc aca 1635

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr

530 535 540

atc aac aga aac atc atg ccg att tgt cta cca aga aaa gaa gct gca 1683

Ile Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala

545 550 555

tcc tta atg aaa aca gac ttc gtt gga act gtg gct ggc tgg ggg tta 1731

Ser Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu

560 565 570

acc cag aag ggg ttt ctt gct aga aac cta atg ttt gtg gac ata cca 1779

Thr Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro

575 580 585 590

att gtt gac cac caa aaa tgt gct act gcg tat aca aag cag ccc tac 1827

Ile Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr

595 600 605

cca gga gca aaa gtg act gtt aac atg ctc tgt gct ggc cta gac cgc 1875

Pro Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg

610 615 620

ggt ggc aag gac agc tgc aga ggt gac agc gga ggg gca tta gtg ttt 1923

Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe

625 630 635

cta gac aat gaa aca cag aga tgg ttt gtg gga gga ata gtt tcc tgg 1971

Leu Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp

640 645 650

ggt tct att aac tgt ggg ggg tca gaa cag tat ggg gtc tac acg aaa 2019

Gly Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys

655 660 665 670

gtc acg aac tat att ccc tgg att gag aac ata ata aat aat ttc taa 2067

Val Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

675 680 685

tttgcaaaaa aaaaaaaaaa aaaa 2091

<210> 54

<211> 685

<212> PRT

<213> rat

<400> 54

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val Ala Thr

1 5 10 15

Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser

20 25 30

Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Thr

65 70 75 80

Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser

100 105 110

Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Thr

130 135 140

Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu

145 150 155 160

Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln Asn

165 170 175

Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg

180 185 190

Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu

195 200 205

Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile Thr

210 215 220

Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala Gln

225 230 235 240

Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr Gly

245 250 255

Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser Asn

260 265 270

Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr Gly

275 280 285

Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro Thr

290 295 300

Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val Leu

305 310 315 320

Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln

325 330 335

Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly

340 345 350

Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly Pro

355 360 365

Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Glu

370 375 380

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe

385 390 395 400

Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe

405 410 415

Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro Val

420 425 430

Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly Gln

435 440 445

Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Glu

450 455 460

Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr Ala

465 470 475 480

Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp Ile

485 490 495

Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala Trp

500 505 510

Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly Phe

515 520 525

Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile Asn

530 535 540

Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu

545 550 555 560

Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr Gln

565 570 575

Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile Val

580 585 590

Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro Gly

595 600 605

Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly Gly

610 615 620

Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp

625 630 635 640

Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser

645 650 655

Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val Thr

660 665 670

Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

675 680 685

<210> 55

<211> 670

<212> PRT

<213> rat

<400> 55

Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val

1 5 10 15

Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp

20 25 30

Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr

65 70 75 80

Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro

85 90 95

Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg

115 120 125

Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr

130 135 140

Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln

145 150 155 160

Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly

165 170 175

Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys

180 185 190

Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile

195 200 205

Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala

210 215 220

Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr

225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser

245 250 255

Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr

260 265 270

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro

275 280 285

Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val

290 295 300

Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu

305 310 315 320

Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp

325 330 335

Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly

340 345 350

Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro

355 360 365

Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr

370 375 380

Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly

385 390 395 400

Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro

405 410 415

Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly

420 425 430

Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly

435 440 445

Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr

450 455 460

Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp

465 470 475 480

Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala

485 490 495

Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly

500 505 510

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile

515 520 525

Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser

530 535 540

Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr

545 550 555 560

Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile

565 570 575

Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro

580 585 590

Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly

595 600 605

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu

610 615 620

Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly

625 630 635 640

Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val

645 650 655

Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

660 665 670

<210> 56

<211> 28

<212> DNA

<213> artificial sequence

<220>

<223> Chinesian

<400> 56

atgaggctgc tgaccctcct gggccttc 28

<210> 57

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Chinesian

<400> 57

gtgcccctcc tgcgtcacct ctg 23

<210> 58

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Chinesian

<400> 58

cagaggtgac gcaggagggg cac 23

<210> 59

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> Chinesian

<400> 59

ttaaaatcac taattatgtt ctcgatc 27

<210> 60

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> mouse

<400> 60

atgaggctac tcatcttcct gg 22

<210> 61

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> mouse

<400> 61

ctgcagaggt gacgcagggg ggg 23

<210> 62

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> mouse

<400> 62

ccccccctgc gtcacctctg cag 23

<210> 63

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> mouse

<400> 63

ttagaaatta cttattatgt tctcaatcc 29

<210> 64

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> rat

<400> 64

gaggtgacgc aggaggggca ttagtgttt 29

<210> 65

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> rat

<400> 65

ctagaaacac taatgcccct cctgcgtcac ctctgca 37

<210> 66

<211> 354

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 66

caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac cctcacgctg 60

acctgcaccg tctctgggtt ctcactcagc aggggtaaaa tgggtgtgag ctggatccgt 120

cagcccccag ggaaggccct ggagtggctt gcacacattt tttcgagtga cgaaaaatcc 180

tacaggacat cgctgaagag caggctcacc atctccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggaccctgtg gacacagcca cgtattactg tgcacggata 300

cgacgtggag gaattgacta ctggggccag ggaaccctgg tcactgtctc ctca 354

<210> 67

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 67

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 68

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 68

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Thr

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 69

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 69

Gln Pro Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr

35 40 45

Gln Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 70

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 70

tcctatgagc tgatacagcc accctcggtg tcagtggccc caggacagac ggccaccatt 60

acctgtgcgg gagacaacct tgggaagaaa cgtgtgcact ggtaccagca gaggccaggc 120

caggcccctg tgttggtcat ctatgatgat agcgaccggc cctcagggat ccctgaccga 180

ttctctgcct ccaactctgg gaacacggcc accctgacca tcactagggg cgaagccggg 240

gatgaggccg actattattg tcaggtgtgg gacattgcta ctgatcatgt ggtcttcggc 300

ggagggacca agctcaccgt ccta 324

<210> 71

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 71

Ser Tyr Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Thr Ile Thr Cys Ala Gly Asp Asn Leu Gly Lys Lys Arg Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Gly Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ala Thr Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly

100 105 110

Ser Glu Gln Lys Leu Ile Ser Glu

115 120

<210> 72

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 72

Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn

1 5 10 15

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu

20 25 30

Trp Cys Asn Gln

35

<210> 73

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 73

Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg

1 5 10 15

Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn

20 25 30

Gln

<210> 74

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 74

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu

1 5 10 15

Trp Cys Asn Gln

20

<210> 75

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 75

Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn

1 5 10 15

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu

20 25 30

Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Gln Val

35 40 45

Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr Leu

50 55 60

Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly Lys Met

65 70 75 80

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

85 90 95

Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser Leu Lys

100 105 110

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

115 120 125

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

130 135 140

Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

145 150 155 160

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

165 170 175

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

180 185 190

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

195 200 205

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

210 215 220

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

225 230 235 240

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

245 250 255

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

260 265 270

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

275 280 285

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

290 295 300

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

305 310 315 320

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

325 330 335

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

340 345 350

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

355 360 365

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

370 375 380

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

385 390 395 400

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

405 410 415

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

420 425 430

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

435 440 445

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

450 455 460

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

465 470 475 480

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490

<210> 76

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 76

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ala Gly

435 440 445

Gly Ser Gly Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp

450 455 460

Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys

465 470 475 480

Thr Lys Leu Trp Cys Asn Gln Gly Ser Gly Ala

485 490

<210> 77

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 77

Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn

1 5 10 15

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu

20 25 30

Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Gln Pro

35 40 45

Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln Thr Ala

50 55 60

Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala Tyr Trp

65 70 75 80

Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr Gln Asp

85 90 95

Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

100 105 110

Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu

115 120 125

Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val Phe Gly

130 135 140

Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser

145 150 155 160

Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala

165 170 175

Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val

180 185 190

Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr

195 200 205

Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu

210 215 220

Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln

225 230 235 240

Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu

245 250 255

Cys Ser

<210> 78

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 78

Gln Pro Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr

35 40 45

Gln Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

115 120 125

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

130 135 140

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

145 150 155 160

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

195 200 205

Thr Glu Cys Ser Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys Glu

210 215 220

Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser

225 230 235 240

Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Gly Ser

245 250 255

Gly Ala

<210> 79

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 79

Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser

1 5 10

<210> 80

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 80

Ala Ala Gly Gly Ser Gly

1 5

<210> 81

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 81

Gly Ser Gly Ala

1

<210> 82

<211> 1533

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 82

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggccttg 60

gaagtgacgt gtgagcccgg aacgacattc aaagacaagt gcaatacttg tcggtgcggt 120

tcagatggga aatcggcggt ctgcacaaag ctctggtgta accagggcac cggtggaggg 180

tcgggatcca gctcacaggt caccttgaag gagtctggtc ctgtgctggt gaaacccaca 240

gagaccctca cgctgacctg caccgtctct gggttctcac tcagcagggg taaaatgggt 300

gtgagctgga tccgtcagcc cccagggaag gccctggagt ggcttgcaca cattttttcg 360

agtgacgaaa aatcctacag gacatcgctg aagagcaggc tcaccatctc caaggacacc 420

tccaaaaacc aggtggtcct tacaatgacc aacatggacc ctgtggacac agccacgtat 480

tactgtgcac ggatacgacg tggaggaatt gactactggg gccagggaac cctggtcact 540

gtctcctcag cctccaccaa gggcccatcc gtcttccccc tggcgccctg ctccaggagc 600

acctccgaga gcacagccgc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 660

acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 720

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 780

acgaagacct acacctgcaa cgtagatcac aagcccagca acaccaaggt ggacaagaga 840

gttgagtcca aatatggtcc cccatgccca ccatgcccag cacctgagtt cctgggggga 900

ccatcagtct tcctgttccc cccaaaaccc aaggacactc tcatgatctc ccggacccct 960

gaggtcacgt gcgtggtggt ggacgtgagc caggaagacc ccgaggtcca gttcaactgg 1020

tacgtggatg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagttcaac 1080

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaacggcaag 1140

gagtacaagt gcaaggtctc caacaaaggc ctcccgtcct ccatcgagaa aaccatctcc 1200

aaagccaaag ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag 1260

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 1320

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1380

ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 1440

caggagggga atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 1500

cagaagagcc tctccctgtc tctcgggaaa tga 1533

<210> 83

<211> 1533

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 83

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtcaccttga aggagtctgg tcctgtgctg gtgaaaccca cagagaccct cacgctgacc 120

tgcaccgtct ctgggttctc actcagcagg ggtaaaatgg gtgtgagctg gatccgtcag 180

cccccaggga aggccctgga gtggcttgca cacatttttt cgagtgacga aaaatcctac 240

aggacatcgc tgaagagcag gctcaccatc tccaaggaca cctccaaaaa ccaggtggtc 300

cttacaatga ccaacatgga ccctgtggac acagccacgt attactgtgc acggatacga 360

cgtggaggaa ttgactactg gggccaggga accctggtca ctgtctcctc agcctccacc 420

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720

cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 840

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1320

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1380

tctctcggga aagccgctgg tggtagtggt ttggaagtga cgtgtgagcc cggaacgaca 1440

ttcaaagaca agtgcaatac ttgtcggtgc ggttcagatg ggaaatcggc ggtctgcaca 1500

aagctctggt gtaaccaggg tagtggtgct tga 1533

<210> 84

<211> 834

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 84

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggccttg 60

gaagtgacgt gtgagcccgg aacgacattc aaagacaagt gcaatacttg tcggtgcggt 120

tcagatggga aatcggcggt ctgcacaaag ctctggtgta accagggcac cggtggaggg 180

tcgggatcca gctcacagcc agtgctgact cagcccccct cactgtccgt gtccccagga 240

cagacagcca gcatcacctg ctctggagag aaattggggg ataaatatgc ttactggtat 300

cagcagaagc caggccagtc ccctgtgttg gtcatgtatc aagataaaca gcggccctca 360

gggatccctg agcgattctc tggctccaac tctgggaaca cagccactct gaccatcagc 420

gggacccagg ctatggatga ggctgactat tactgtcagg cgtgggacag cagcactgcg 480

gtattcggcg gagggaccaa gctgaccgtc ctaggccagc ctaaggcggc gccctcggtc 540

accctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 600

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 660

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 720

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 780

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atag 834

<210> 85

<211> 834

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 85

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

ccagtgctga ctcagccccc ctcactgtcc gtgtccccag gacagacagc cagcatcacc 120

tgctctggag agaaattggg ggataaatat gcttactggt atcagcagaa gccaggccag 180

tcccctgtgt tggtcatgta tcaagataaa cagcggccct cagggatccc tgagcgattc 240

tctggctcca actctgggaa cacagccact ctgaccatca gcgggaccca ggctatggat 300

gaggctgact attactgtca ggcgtgggac agcagcactg cggtattcgg cggagggacc 360

aagctgaccg tcctaggcca gcctaaggcg gcgccctcgg tcaccctgtt cccgccctcc 420

tctgaggagc ttcaagccaa caaggccaca ctggtgtgtc tcataagtga cttctacccg 480

ggagccgtga cagtggcctg gaaggcagat agcagccccg tcaaggcggg agtggagacc 540

accacaccct ccaaacaaag caacaacaag tacgcggcca gcagctatct gagcctgacg 600

cctgagcagt ggaagtccca cagaagctac agctgccagg tcacgcatga agggagcacc 660

gtggagaaga cagtggcccc tacagaatgt tcagccgctg gtggtagtgg tttggaagtg 720

acgtgtgagc ccggaacgac attcaaagac aagtgcaata cttgtcggtg cggttcagat 780

gggaaatcgg cggtctgcac aaagctctgg tgtaaccagg gtagtggtgc ttag 834

<210> 86

<211> 500

<212> PRT

<213> Chile person

<400> 86

Met Ala Ser Arg Leu Thr Leu Leu Thr Leu Leu Leu Leu Leu Leu Ala

1 5 10 15

Gly Asp Arg Ala Ser Ser Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln

20 25 30

Asp Pro Glu Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr

35 40 45

Val Ile Ser Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser

50 55 60

Leu Pro Thr Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala

65 70 75 80

Asn Thr Thr Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr

85 90 95

Gln Pro Thr Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp

100 105 110

Ser Pro Thr Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr

115 120 125

Leu Cys Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp

130 135 140

Ala Leu Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met

145 150 155 160

Lys Lys Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser

165 170 175

Leu Leu Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn

180 185 190

Leu Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln

195 200 205

Ala Leu Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile

210 215 220

Phe His Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser

225 230 235 240

Arg Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp

245 250 255

Ala Asn Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn

260 265 270

Lys Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val

275 280 285

Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp

290 295 300

Pro Lys Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile

305 310 315 320

Lys Val Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile

325 330 335

Asp Gln Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn

340 345 350

Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu

355 360 365

Asp Met Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu

370 375 380

Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg

385 390 395 400

Ile Lys Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu

405 410 415

Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu

420 425 430

Asp Pro Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu

435 440 445

Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val

450 455 460

Ala Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val

465 470 475 480

Leu Trp Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr

485 490 495

Asp Pro Arg Ala

500

<210> 87

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 87

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Leu Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Gly Asn Thr Tyr His Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg His Gly Asp Phe Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 88

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 88

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg

<210> 89

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 89

Ser Tyr Leu Met Ser

1 5

<210> 90

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 90

Thr Ile Ser Gly Gly Gly Gly Asn Thr Tyr His Pro Asp Ser Met Lys

1 5 10 15

Gly

<210> 91

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 91

His Gly Asp Phe Gly Asn Tyr Phe Asp Tyr

1 5 10

<210> 92

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 92

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Thr Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 93

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 93

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 94

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 94

Lys Gln Ser Tyr Asn Leu Phe Thr

1 5

<210> 95

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 95

gaggtgaagc tggtggagtc tgggggaggc ttggtgaagc ctggagggtc cctaaaactc 60

tcctgtgcag cctcaggatt cactttcagt agttatctta tgtcttgggt tcgccagact 120

ccggagaaga ggctggagtg ggtcgcaacc attagtggtg gtggtggtaa cacttaccat 180

ccagacagta tgaagggtcg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgag gtctgaggac acggccttgt attactgtgc aagacatggg 300

gactttggta actacttcga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210> 96

<211> 339

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 96

gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcgggaga gaaggtcact 60

atgagctgca aatccagtca gagtctgctc aacagtggaa cccaaaagaa ctacttggct 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 300

ttcacgttcg gtgctgggac caagctggag ctgaaacgg 339

<210> 97

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 97

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Thr

20 25 30

Tyr Trp Gly Val Thr Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 98

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic

<400> 98

Gln Pro Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr

35 40 45

Gln Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

Claims

1. Methods for treating, inhibiting, reducing or preventing acute respiratory distress syndrome, pneumonia or some other pulmonary or other acute manifestations of covd-19, such as thrombosis, in a mammalian subject infected with SARS-CoV-2 comprising

(ii) Administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation to a subject having an increased level of the MASP-2/C1-INH complex, optionally wherein the amount of the MASP-2 inhibitor is sufficient to reduce the level of MASP-2/C1-INH to a control level or reference standard.

2. The method of claim 1, wherein the MASP-2 inhibitor is a MASP-2 antibody or fragment thereof.

3. The method of claim 2, wherein the MASP-2 inhibitor specifically binds to SEQ ID NO:6 or a fragment thereof.

4. The method of claim 2, wherein the MASP-2 antibody or fragment thereof specifically binds with an affinity that is at least 10-fold greater than it binds a different antigen in the complement system comprising SEQ ID NO: 6.

5. The method of claim 2, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

6. The method of claim 1, wherein the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1 q-dependent complement activation.

7. The method of claim 1, wherein the MASP-2 inhibitor is a small molecule MASP-2 inhibitory compound.

8. The method of claim 7, wherein the MASP-2 inhibitory compound is a synthetic or semi-synthetic small molecule.

9. The method of claim 1, wherein the inhibitor of MASP-2 is an inhibitor of expression of MASP-2.

10. The method of claim 1, wherein the MASP-2 inhibitor is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, orally, or as an inhalant.

11. The method of claim 2, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67, CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence depicted in SEQ ID NO:69, CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence depicted.

12. The method of claim 2, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:67, said light chain variable region comprises SEQ ID NO:69.

13. a method for treating, reducing, preventing or reducing the risk of developing one or more of the long-term sequelae associated with COVID-19 in a mammalian subject having been infected with SARS-CoV-2, comprising

14. The method of claim 13, wherein the MASP-2 inhibitor is a MASP-2 antibody or fragment thereof.

15. The method of claim 14, wherein the MASP-2 inhibitor specifically binds to SEQ ID NO:6 or a fragment thereof.

16. The method of claim 14, wherein the MASP-2 antibody or fragment thereof specifically binds with an affinity that is at least 10-fold greater than it binds a different antigen in the complement system comprising SEQ ID NO: 6.

17. The method of claim 14, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

18. The method of claim 14, wherein the MASP-2 inhibitor selectively inhibits lectin pathway complement activation without substantially inhibiting C1 q-dependent complement activation.

19. The method of claim 13, wherein the MASP-2 inhibitor is a small molecule MASP-2 inhibitory compound.

20. The method of claim 13, wherein the MASP-2 inhibitory compound is a synthetic or semi-synthetic small molecule.

21. The method of claim 13, wherein the inhibitor of MASP-2 is an inhibitor of expression of MASP-2.

22. The method of claim 14, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67, and CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:69, CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence depicted.

23. The method of claim 14, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:67, said light chain variable region comprises SEQ ID NO:69.

24. the method of claim 13, wherein the one or more covd-19 associated long-term sequelae is selected from cardiovascular complications (including myocardial injury, cardiomyopathy, myocarditis, intravascular coagulation, stroke, venous and arterial complications, and pulmonary embolism); neurological complications (including cognitive difficulties, confusion, memory loss also known as "brain fog", headache, stroke, dizziness, syncope, seizures, anorexia, insomnia, olfactory loss, gustatory loss, myoclonus, neuropathic pain, myalgia, neurological diseases such as alzheimer's disease, guillain-barre syndrome, miller-fisher syndrome, the development of parkinson's disease, kidney damage (e.g., acute Kidney Injury (AKI)), pulmonary complications (including pulmonary fibrosis, dyspnea, pulmonary embolism), inflammatory conditions such as kawasaki disease, kawasaki-like disease, childhood multisystem inflammatory syndrome, multisystem organ failure, extreme fatigue, muscle weakness, low fever, inattention, memory errors, mood changes, sleep difficulties, needle pain in arms and legs, diarrhea and vomiting, loss of taste and smell, sore throat and dysphagia, new episodes of diabetes and hypertension, rash, shortness of breath, chest pain and palpitations.

25. A monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human MASP-2 complexed with C1-INH, wherein the antibody comprises a binding domain comprising (a) a polypeptide as set forth in SEQ ID NO:87 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:88, or (b) LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:97 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:98, LC-CDR1, LC-CDR2 and LC-CDR3 in the light chain variable region,

wherein the CDRs are numbered according to the Kabat numbering system.

26. The monoclonal antibody of claim 25, wherein the antibody comprises a sequence identical to the sequence set forth in SEQ ID NO:87 and a heavy chain variable region having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:88, a light chain variable region having at least 95% identity to the amino acid sequence depicted; or wherein the antibody comprises a sequence identical to the sequence set forth in SEQ ID NO:97 and a heavy chain variable region having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:98, and a light chain variable region having at least 95% identity to the amino acid sequence set forth in seq id no.

27. The monoclonal antibody of claim 25, wherein the antibody is a humanized antibody, chimeric antibody, or fully human antibody.

28. A method of measuring the amount of MASP-2/C1-INH in a biological sample comprising:

(a) Providing a test biological sample from a human subject;

29. The method of claim 28, wherein the biological sample is a fluid sample selected from the group consisting of whole blood, serum, plasma, urine, and cerebrospinal fluid.

30. The method of claim 28, wherein the antibody that specifically binds MASP-2 comprises a binding domain comprising (a) a polypeptide as set forth in SEQ ID NO:87 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:88, or (b) LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region as set forth in SEQ ID NO:97 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

31. The method of claim 28, wherein the human subject is currently infected with SARS-CoV-2, or has been previously infected with SARS-CoV-2, or wherein the subject has or is at risk of developing another lectin pathway disease or condition (e.g., HSCT-TMA, igAN, gvHD or other lectin pathway disease or disorder).

32. A method of determining the risk of a subject infected with SARS-CoV-2 or having been infected with SARS-CoV-2 to develop a covd-19-associated ARDS or other adverse outcome or long-term sequelae associated with covd-19, comprising:

(a) Obtaining a biological sample from the subject;

(b) Measuring the level of MASP-2/C1-INH complex in the sample;

(c) Comparing the measured level with a predetermined level of MASP-2/C1-INH complex or a reference standard to assess the risk of developing ARDS associated with COVID-19 and/or long-term sequelae associated with COVID-19; and

(d) Determining the risk of the subject developing a covd-19 related ARDS or other adverse outcome and/or long-term sequelae associated with covd-19 and reporting the outcome to a patient, physician, or database;

(e) Optionally, the treatment is administered to a subject determined to be likely to develop an acute disease and/or long-term sequelae associated with a covd-19 infection.

33. The method of claim 32, wherein the level of MASP-2/C1-INH complex is measured in an immunoassay.

34. The method of claim 32, wherein the method comprises performing an immunoassay to measure the level of MASP-2/C1-INH complex in the biological sample.

35. The method of claim 34, wherein the immunoassay is an ELISA assay.

36. The method of claim 35, wherein the immunoassay comprises the use of a capture antibody that specifically binds MASP-2, the capture antibody comprising a binding domain comprising the amino acid sequence set forth in SEQ ID NO:87 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:88, wherein the CDRs are numbered according to the Kabat numbering system, LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region.

37. The method of claim 34, wherein the immunoassay is a bead-based immunofluorescent assay.

38. The method of claim 37, wherein the immunoassay comprises the use of a capture antibody that specifically binds MASP-2, the capture antibody comprising a binding domain comprising the amino acid sequence as set forth in SEQ ID NO:97 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

39. The method of claim 37 or 38, wherein the biological sample is serum or plasma.

40. The method of claim 39, wherein the biological sample is 1% to 5% serum or plasma.

41. A method for monitoring the efficacy of treatment with a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, in a mammalian subject in need thereof, the method comprising:

42. The method of claim 41, wherein the method further comprises adjusting the dosage of the MASP-2 inhibitory antibody or antigen binding fragment thereof.

43. The method of claim 42, wherein the dose of MASP-2 inhibitory antibody or antigen binding fragment thereof administered to the subject is increased if the level of MASP-2/C1-INH complex is higher than a control or reference standard.

44. The method of claim 43, wherein if an increased dose of MASP-2 inhibitory antibody or antigen binding fragment thereof is administered to the subject, steps (b) through (e) are repeated to determine if the increased dose is sufficient to adjust the level of MASP-2/C1-INH complex to the desired level as compared to the respective control or reference standard.

45. The method of claim 41, wherein steps (b) and (d) comprise assessing the concentration of MASP-2/CI-INH complex in said biological sample in an immunoassay.

46. The method of claim 45, wherein the immunoassay is a bead-based immunofluorescent assay.

47. The method of claim 46, wherein the immunoassay comprises the use of a capture antibody that specifically binds MASP-2, the capture antibody comprising a binding domain comprising the amino acid sequence set forth in SEQ ID NO:97 and comprising HC-CDR1, HC-CDR2 and HC-CDR3 in the heavy chain variable region as set forth in SEQ ID NO:98, wherein the CDRs are numbered according to the Kabat numbering system.

48. The method of claim 46 or 47, wherein the biological sample is serum or plasma.

49. The method of claim 48, wherein the biological sample is 1% to 5% serum or plasma.

50. The method of any one of claims 41-50, wherein the mammalian subject is a human subject.

51. The method of claim 50, wherein the human subject has or is at risk of developing a lectin pathway disease or disorder selected from the group consisting of HSCT-TMA, igAN, lupus nephritis, and graft versus host disease or some other lectin pathway disease or disorder.

52. The method of claim 41, wherein the human subject has or is at risk of developing COVID-19 or a long-term sequelae associated with COVID-19.

53. The method of claim 41, wherein the second time point is 2 to 14 days after the first time point.

54. The method of claim 41, wherein the second time point is within 2 to 7 days from the first time point.

55. The method of claim 41, wherein the second time point is within 2 to 4 days from the first time point.