EA043102B1 - METHOD FOR REDUCING PROTEINURIA IN A HUMAN SUFFERING FROM IMMUNOGLOBULIN A NEPHROPATHY - Google Patents

Description

Sequence List Statement

The sequence listing associated with this application is presented in text format instead of a paper copy and is incorporated into this application by reference. The text file containing the list of sequences is named MP_l_0269_PCT_Sequence_Listing_20171013_ST25. The text file is 136 KB in size and was created on October 10, 2017 and has been available via EFS-Web since the description was submitted.

State of the art

In humans and other vertebrates, the complement system provides an early action mechanism to initiate, amplify, and regulate the immune response to microbial infection and other acute infections (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 an important first line of defense against potential pathogens, complement activity that stimulates a protective immune response may also pose a potential threat to the host (K. R. Kalli et al., Springer Semin. Immunopathol., 15:41-431, 1994; B. P. Morgan, Eur. J. Clinical Investig., 24:219-228, 1994). For example, the proteolytic products C3 and C5 stimulate neutrophil recruitment and activation. Although neutrophils are essential for host defense, activated neutrophils are not selective in their release of destructive enzymes and can cause organ damage. In addition, complement activation can cause the deposition of lytic complement components on adjacent host cells as well as on microbial targets, leading to host cell lysis.

The complement system is also involved in the pathogenesis of numerous acute and chronic diseases, including myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leakage after thermal burns, inflammation after cardiopulmonary bypass surgery, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement itself does not cause these conditions, but is one of several factors involved in pathogenesis. Nevertheless, complement activation may be the underlying pathological mechanism and represents an effective link for clinical control in many of these disease states. The increasing recognition of the importance in understanding the mechanisms of complement-mediated tissue damage in various disease states further highlights the need for the development of effective complement-inhibiting drugs. To date, Eculizumab (Solaris® (Solaris)), i.e. anti-C5 antibody is the only complement-targeting drug approved for human use. However, C5 is one of several effector molecules that act in the late stages of the complement system, and blockade of C5 does not result in suppression of complement system activation. Therefore, an inhibitor of the initiation steps of complement activation would have a significant advantage over a complement component inhibitor acting at later stages.

It is now generally accepted that the complement system can be activated in three different ways: classical, lectin and alternative. The classical pathway is usually triggered by a complex consisting of host antibodies bound to a foreign particle (ie, antigen) and therefore requires prior exposure to the antigen in order to generate a specific antibody response. Since the activation of the classical pathway depends on the host's prior adaptive immune response, the classical pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

Activation of the complement system leads to sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of the C1q specific recognition molecule to antigen-bound IgG and IgM molecules. C1q is associated with the serine protease proenzymes C1r and C1s in a complex called C1. Upon binding of C1q to the immune complex, autoproteolytic Arg-Ile cleavage of the C1r site is followed by C1r-mediated cleavage and activation of C1s, thus providing the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and C2 is similarly cleaved into C2a and C2b. C4b fragments can form covalent bonds with adjacent hydroxyl groups or amino groups and generate C3 convertase (C4b2a) via non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 through proteolytic cleavage into subcomponents C3a and C3b, leading to the formation of C5 convertase (C4b2a3b), which through cleavage of C5 leads to the formation of a membrane attack complex (C5b together with C6, C7, C8 and C9, also called MAC), which can destroy cell membranes, leading to cell lysis. Activated forms of C3 and C4 (C3b and C4b) are covalently deposited on foreign target surfaces, which are recognized by complement receptors on many phagocytes.

Regardless of this, the first step in the activation of the complement system via the lectin pathway is also the binding of specific recognition molecules, after which activation occurs.

- 1 043102 proenzymes associated with serine protease. However, instead of binding immune complexes via C1q, recognition molecules in the lectin pathway contain a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL11) collectively known as lectins. See J. Lu et al., Biochim. Biophys. Acta, 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol., 21:547-578 (2003); Teh et al., Immunology, 101:225-232 (2000)). See also J. Luet et al., Biochim. Biophys. Acta, 1572:387-400 (2002); Holmskov et al., Annu. Rev. Immunol., 21:547-578 (2003); Teh et al., Immunology, 101:225-232 (2000); Hansen et al., J. Immunol., 185(10):6096-6104 (2010).

Ikeda et al. demonstrated for the first time that MBL, like C1q, can activate the complement system after binding to yeast mannan-coated erythrocytes in a C4-dependent mechanism (Ikeda et al., J. Biol. Chem., 2(52):7451-7454, ( 1987)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates at 3- and 4-hydroxy groups oriented in the horizontal plane of the pyranose ring. Thus, the main ligands for MBL are D-mannose and N-acetyl-D-glucosamine, and carbohydrates that do not meet this steric requirement have an undetectable affinity for MBL (Weis et al., Nature, 360:127-134 (1992) ). The interaction between MBL and monovalent sugars is very weak with dissociation constants typically in the single digit millimolar range. Strong, specific binding of MBL to glycan ligands is achieved through avidity, i. interacting simultaneously with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys., 299:129-136 (1992)). MBL recognizes carbohydrate patterns typically produced by microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars typically found in mature complex glycoconjugates present on the cell surface and in mammalian plasma. This binding specificity is thought to promote recognition of foreign surfaces and help protect against autoactivation. However, MBL does not bind with high affinity to high-mannose glycan precursor clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and the Golgi apparatus of mammalian cells (Maynard et al., J. Biol. Chem., 257:3788- 3794 (1982)). Thus, damaged cells are potential targets for activation of the lectin pathway through MBL binding.

Ficolins contain a lectin domain that is different from the MBL lectin domain, called the fibrinogen-like domain. Ficolins bind to sugar residues in a Ca~-independent manner. Three types of ficolins have been identified in humans (L-ficolin, M-ficolin, and H-ficolin). Two serum ficolins, L-ficolin and H-ficolin, share a common specificity for N-acetyl-D-glucosamine, but H-ficolin also binds to N-acetyl-D-galactosamine. The difference in sugar specificity between L-ficolin, H-ficolin, CL-11, and MBL means that different lectins can be complementary and act on different, even overlapping, glycoconjugates. This concept is supported by recent reports in the literature that of the known lectins in the lectin pathway, only L-ficolin specifically binds to lipoteichoic acid, i.e. cell wall glycoconjugate found in all Gram-positive bacteria (Lynch, N.J. et al., J. Immunol., 172:1198-1202, 2004). Collectins (ie MBL) and ficolins do not have significant amino acid sequence similarity. However, these two groups of proteins have the same domain organization and, like C1q, assemble into oligomeric structures, which maximizes the possibility of multisite binding.

Serum MBL concentrations are highly variable in populations of healthy individuals and are genetically controlled by polymorphisms/mutations in the promoter and coding regions of the MBL gene. Expression of MBL, as an acute phase protein, is further enhanced during inflammation. L-ficolin is present in serum at concentrations similar to MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable in strength to the MBL branch. MBL and ficolins also function as opsonins, allowing phagocytes to act on MBL- and ficolin-decorated surfaces (see Jack et al., J. Leukoc. Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002) Aoyagi et al., J. Immunol., 174(1):418-25 (2005) Such opsonization requires interaction of these proteins with phagocyte receptors (Kuhlman et al., J. Exp. Med., 169:1733 (1989) Matsushita et al., J. Biol. Chem., 277:2448-54 (1996)), whose identity has not yet been established.

The specific and high-affinity interaction of human MBL with unique C1r/C1s-like serine proteases called MBL-associated serine proteases (MASPs) is mediated through its collagen-like domain. So far, three MASPs have been described. First, one MASP enzyme was identified and characterized as the enzyme responsible for initiating the complement cascade (i.e., C2 and C4 cleavage) (Matsushita et al., J. Exp. Med., 176(6):1497-1502 ( 1992); Ji et al., J. Immunol., 750:571-578 (1993)). It was then determined that MASP activity is actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature, 556:506-510 (1997)). However, it has been shown that one is enough to activate complement.

- 2 043102 MBL-MASP-2 complex (Vorup-Jensen et al., J. Immunol., 165:2093-2100 (2000)). In addition, only MASP-2 cleaved C2 and C4 at a high rate (Ambrus et al., J. Immunol., 770:1374-1382 (2003)). Thus, MASP-2 is the protease responsible for the activation of C4 and C2 to form a C3 convertase, C4b2a. This is a significant difference from the C1 complex of the classical pathway, where the coordinated action of two specific serine proteases (C1r and C1s) leads to the activation of the complement system. In addition, a third new protease, MASP-3, has been isolated (Dahl, M.R. et al., Immunity, 15:127-35, 2001). MASP-1 and MASP-3 are alternative splicing products of the same gene.

The organization of MASP domains is identical to that of C1r and C1s, which are enzymatic components of the C1 complex (Sim et al., Biochem. Soc. Trans., 28:545 (2000)). These domains include the sea urchin N-terminal C1r/C1s/VEGF/bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement regulatory protein domains, and a serine protease domain. As in C1 proteases, activation of MASP-2 is mediated through cleavage of the Arg-Ile bond adjacent to the serine protease domain, leading to separation of the enzyme into A and B chains connected by a disulfide bond, where the latter consists of the serine protease domain.

MBL can also be associated with an alternatively spliced form of MASP-2, known as 19 kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP-2 (Stover, J. Immunol. , 162:3481-90, (1999); Takahashi et al., Int. Immunol., 11:859-863, (1999)). MAp19 contains the first two MASP-2 domains followed by an additional sequence of four unique amino acids. The function of MAp19 remains unclear (Degn et al., J. Immunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology, 205:455-466 (2002)).

There is some evidence to suggest that various MBL-MASP complexes and a large fraction of serum MASP do not complex with MBL (Thiel, S. et al., J. Immunol., 165:878-887, 2000). Both H- and L-ficolin bind to all MASPs and, like MBL, activate the complement lectin pathway (Dahl, M.R. et al., Immunity, 15:127-35, 2001; Matsushita, M. et al., J Immunol., 165:3502-3506, 2002). The lectin and classical pathways form a common C3 convertase (C4b2a), at which point the two pathways converge.

There is a widespread belief that the lectin pathway plays an important role in protecting the uninfected host from infection. Strong evidence for the involvement of MBL in host defense has been obtained from an analysis of patients with reduced serum levels of functional MBL (Kilpatrick, D.C., Biochim. Biophys. Acta, 1572:401-413, 2002). These patients are susceptible to recurrent bacterial and fungal infections. Such symptoms usually appear early in life, within the apparent window of vulnerability, when maternal antibody titers decline until a full antibody repertoire is formed. This syndrome is often the result of mutations at several sites in the MBL collagen portion that prevent proper formation of MBL oligomers. However, since MBL can function as an opsonin independent of complement, it is not known to what extent the increased susceptibility to infection is due to decreased complement activation.

All three pathways (i.e. classical, lectin and alternative) are thought to converge at C5, which is cleaved to form products with multiple pro-inflammatory effects. The pathway where all three pathways converge has been defined as the terminal complement pathway. C5a is the most potent anaphylotoxin, inducing changes in smooth muscle and vascular tone, as well as vascular permeability. It is also a potent chemotaxin and an activator of neutrophils and monocytes. C5a-mediated cell activation can significantly enhance inflammatory responses by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 results in the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublithic MAC deposition, in addition to its role as a lytic pore-forming complex, may play an important role in the inflammatory process.

In addition to its important role in immune defense, the complement system is involved in tissue damage in many clinical conditions. Despite the existence of a wealth of data indicating the involvement of the classical and alternative complement pathways in the pathogenesis of noncommunicable diseases in humans, the role of the lectin pathway in such pathogenesis has only just begun to be assessed. Recent studies have shown that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia/reperfusion injury. Collard et al. (2000) showed that cultured endothelial cells subjected to oxidative stress bind to MBL and exhibit C3 deposition in response to exposure to human serum (Collard et al., Am. J. Pathol., 156:1549-1556 (2000)). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies suppressed MBL binding and complement activation. These data were confirmed in a rat model of myocardial ischemia/reperfusion, in which

- 3043102 rats treated with blocking antibodies to rat MBL showed a significant reduction in myocardial injury due to coronary artery occlusion compared to rats treated with a control antibody (Jordan, J.E. et al., Circulation, 104:1413-1418, 2001). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress remains unclear, but recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins rather than glycoconjugates (Collard, C.D. et al., Am J. Pathol., 159:1045-1054, 2001). Other studies have pointed to the role of the classical and alternative pathways in the pathogenesis of ischemic/reperfusion injury, and the role of the lectin pathway in the pathogenesis of this disease remains a matter of debate (Riedermann, N.C. et al., Am. J. Pathol., 162:363-367, 2003).

Fibrosis is the formation of excess connective tissue in an organ or tissue, usually in response to injury or injury. The hallmark of fibrosis is the production of excess extracellular matrix after local injury. The normal physiological response to injury leads to the formation of connective tissue, but this initially healing process of repair can be delayed and become pathological, altering tissue architecture and function. 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, leads to the release of cytokines and enhances the deposition of collagen, fibronectin, and other molecular markers of fibrosis. Traditional therapeutic approaches have mainly focused on the inflammatory process in fibrosis using corticosteroids and immunosuppressants. Unfortunately, all these anti-inflammatory agents have little clinical effect. There is currently no effective treatment or therapy for fibrosis, but both animal studies and anecdotal reports of human outcomes suggest that fibrotic tissue lesions can be reversed (Tampe and Zeisberg, Nat. Rev. Nephrol., vol. 10 :226-237, 2014).

The kidney has a limited ability to recover from injury. Various pathologies of the kidneys lead to local inflammation, which causes scarring and fibrosis of the kidneys. Continued exposure to inflammatory stimuli leads to tubulointerstitial inflammation and fibrosis, and progressive renal dysfunction in chronic kidney disease. Its progression to end-stage renal disease is associated with significant morbidity and mortality. Because tubulointerstitial fibrosis is a common endpoint for many renal pathologies, it represents an important target for treatment to prevent renal failure. Risk factors (eg, proteinuria) independent of primary kidney disease contribute to renal fibrosis and loss of renal excretory function by stimulating local inflammation, which in turn increases disease progression.

Given the role of fibrosis in many diseases and disorders, such as, for example, tubulointerstitial fibrosis leading to chronic kidney disease, there is an urgent need to develop therapeutically effective agents for the treatment of diseases and conditions caused or complicated by fibrosis. Also given the lack of new and existing therapies that target inflammatory profibrotic factors in renal disease, there is a need to develop therapeutically effective agents for the treatment, suppression, prevention and/or management of renal fibrosis, thereby preventing the progression of chronic kidney disease.

The essence of the invention

This section of the description is intended to introduce a set of concepts presented in a simplified form, which are described in more detail below. This section of the description is not intended to define key features of the claimed subject matter and is not intended to define the scope of the claimed subject matter.

In one aspect, the invention relates to a method of treating, suppressing, alleviating, or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused by or complicated by fibrosis and/or inflammation, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to suppress fibrosis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to part of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits complement lectin pathway activation without suppressing essentially dq-dependent complement activation. In one embodiment, the subject suffers from a disease or disorder caused by or complicated by at least one of the following:

(i) fibrosis and/or inflammation associated with ischemic reperfusion injury;

(ii) fibrosis and/or inflammation of the kidneys (eg, tubulointerstitial fibrosis, chronic

- 4 043102 kidney disease, chronic renal failure, glomerular disease (eg, focal segmental glomerulosclerosis), immune complex disorders (eg, IgA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial lesions, and glomerulonephritis (eg, C3-glomerulopathy);

(iii) fibrosis and/or inflammation of the lungs (eg, chronic obstructive pulmonary disease, cystic fibrosis, scleroderma-associated pulmonary fibrosis, bronchiectasis, and pulmonary hypertension);

(iv) liver fibrosis and/or inflammation (eg, cirrhosis, non-alcoholic fatty liver disease (steatohepatitis)), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to acute or chronic hepatitis (drug-induced hepatotoxicity, caused by acetaminophen or another drug);

(v) fibrosis and/or inflammation of the heart (eg, cardiac fibrosis, myocardial infarction, valvular fibrosis, atrial fibrosis, endomyocardial fibrosis, arrhythmic right ventricular cardiomyopathy (ARCH);

(vi) vascular fibrosis (eg, vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis, and abdominal aortic aneurysm);

(vii) skin fibrosis (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids, connective tissue disease, scarring, and hypertrophic scars);

(viii) fibrosis of the joints (eg, arthrofibrosis);

(ix) fibrosis of the central nervous system (eg, stroke, traumatic brain injury and spinal cord injury);

(x) fibrosis of the digestive system (eg, Crohn's disease, pancreatic fibrosis, and ulcerative colitis);

(xi) ocular fibrosis (eg, anterior subcapsular cataract, posterior capsule opacities, macular degeneration, and retinal and vitreal retinopathy);

(xii) fibrosis of soft tissue structures of the musculoskeletal system (eg, cellular capsulitis, Dupuytren's contracture, and myelofibrosis);

(xiii) fibrosis of the reproductive organs (eg, endometriosis and Peyronie's disease);

(xiv) a chronic infectious disease that causes fibrosis and/or inflammation (eg, alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza);

(xv) an autoimmune disease that causes fibrosis and/or inflammation (eg, scleroderma and systemic lupus erythematosus (SLE));

(xvi) trauma-related scarring (eg, where trauma-related scarring is selected from the group consisting of surgical complications (eg, postoperative adhesions, in which scar tissue can form between internal organs, causing contracture, pain, and can lead to infertility) , chemotherapy-induced fibrosis, radiation-induced fibrosis, and scarring associated with burns); or (xvii) organ transplant, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, and pleural fibrosis.

In another aspect, the present invention relates to a method of treating, suppressing, alleviating, or preventing fibrosis of the kidneys in a mammal suffering from or at risk of developing a disease or disorder caused by or complicated by fibrosis and/or inflammation of the kidneys, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to suppress renal fibrosis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to part of SEQ ID NO: 6. In one embodiment, the MASP-2 antibody, or fragment thereof, specifically binds to a polypeptide containing SEQ ID NO: 6, with an affinity at least 10 times greater than the binding affinity for another antigen in the complement system. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory agent selectively inhibits the activation of the lectin complement pathway without substantially inhibiting C1q-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, or by inhalation. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to inhibit tubulointerstitial fibrosis. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in a subject. In one embodiment, the subject suffers from a kidney disease or a disease selected from the group consisting of chronic kidney disease, chronic kidney disease, glomerular disease (e.g., focal segmental

- 5 043102 glomerulosclerosis), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial lesions and glomerulonephritis (eg C3 glomerulopathy). In one embodiment, the subject suffers from proteinuria and the MASP-2 inhibitory agent is administered in an amount effective to reduce the severity of the subject's proteinuria. In one embodiment, the MASP-2 inhibitory agent is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction (e.g., at least a 30% reduction, or at least a 40% reduction, or at least a 50% reduction) in 24-hour urinary protein excretion compared to the subject's baseline 24-hour urinary protein excretion prior to treatment. In one embodiment, the subject suffers from a proteinuria-associated kidney disease or disorder selected from the group consisting of nephrotic syndrome, preeclampsia, exlampsia, kidney toxicity, amyloidosis, vascular-collagenous diseases (e.g., systemic lupus erythematosus), dehydration, glomerular diseases (eg, membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy (eg, Berger's disease), IgM- nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics ( eg adriamycin) or opiates (eg heroin) or other nephrotoxins)); Fabry disease, infections (eg, HIV, syphilis, hepatitis A, B, or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), ebola hemorrhagic fever, nail patella syndrome, familial fever, HELLP syndrome, systemic lupus erythematosus, granulomatosis Wegener's disease, rheumatoid arthritis, glycogen storage disease type 1 (Girke's disease), Goodpasture's syndrome, Henoch-Schönlein purpura, urinary tract infection that has spread to the kidneys, Sjögren's syndrome, and post-infectious glomerulonephritis. In one embodiment, the subject is suffering from IgA nephropathy. In one embodiment, the subject suffers from membranous nephropathy.

In another aspect of the present invention, there is provided a method for preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, comprising administering an amount of a MASP-2 inhibitory agent effective to reduce or prevent proteinuria in the subject. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to part of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits complement lectin pathway activation without suppressing essentially the activation of a C1q-dependent complement. In one embodiment, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, exlampsia, kidney toxicity, amyloidosis, vascular collagen diseases (e.g., systemic lupus erythematosus), dehydration, glomerular diseases (e.g., membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy (eg, Berger's disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (eg, adriamycin ) or opiates (e.g. heroin)); Fabry disease, infections (eg, HIV, syphilis, hepatitis A, B, or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), ebola hemorrhagic fever, nail patella syndrome, familial fever, HELLP syndrome, systemic lupus erythematosus, granulomatosis Wegener's disease, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, Schönlein's purpura, Henoch's purpura, urinary tract infection that has spread to the kidneys, Sjögren's syndrome, and postinfectious glomerulonephritis. In one embodiment, the MASP-2 inhibitory agent is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction (e.g., at least 30% reduction or at least 40%

- 6 043102 reduction, or at least 50% reduction) in 24-hour urinary protein excretion compared to the subject's baseline 24-hour urinary protein excretion before treatment.

In another aspect, the present invention provides a method for inhibiting the progression of chronic kidney disease, comprising administering to a subject in need thereof an amount of a MASP-2 inhibitory agent effective to reduce or prevent renal fibrosis, eg, tubulointerstitial fibrosis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to part of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits complement lectin pathway activation without suppressing essentially the activation of a C1q-dependent complement. In one embodiment, the subject in need thereof was suffering from proteinuria prior to the administration of the MASP-2 inhibitory agent, wherein the administration of the MASP-2 inhibitory agent reduces the subject's proteinuria. In one embodiment, the MASP-2 inhibitory agent is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction (e.g., at least a 30% reduction or at least a 40% reduction, or at least 50% reduction) in 24-hour urinary protein excretion compared to baseline 24-hour urinary protein excretion in the subject prior to treatment. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in a subject.

In another aspect of the present invention, a method is provided for protecting the kidneys from kidney damage in a subject who has received, is receiving, or will be receiving treatment with one or more nephrotoxic agents, comprising administering an amount of a MASP-2 inhibitory agent effective to prevent or alleviate drug-induced nephropathy. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to part of SEQ ID NO: 6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits complement lectin pathway activation without suppressing essentially the activation of a C1q-dependent complement.

In one aspect of the invention, a method of treating a human suffering from immunoglobin A nephropathy (IgAN) is provided, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to suppress MASP-2-dependent complement activation. In one embodiment, the subject suffers from steroid dependent IgAN. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit classical pathway activation. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 3 0 nM or less. In one embodiment, the method further comprises identifying a subject having a steroid-dependent IgAN prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve kidney function. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount effective to improve renal function. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion compared to baseline 24-hour protein excretion. in the urine of the subject prior to treatment. In one embodiment, the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in the specified subject. 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 shown in SEQ ID NO: 67, a light chain variable region comprising CDR -L1, CDR-L2 and CDR-L3 amino acid sequence shown in SEQ ID NO: 69.

In another aspect of the invention, a method of treating a human suffering from membranous nephropathy (MN) is provided, comprising administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to suppress MASP-2-dependent complement activation. In one embodiment, the subject suffers from steroid-dependent MN. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve kidney function. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion from baseline 24-hour protein excretion in the urine of the subject prior to treatment. In one embodiment, the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in the specified subject. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDRH2 and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, a light chain variable region comprising CDR-L1 , CDR-L2 and CDR-L3 amino acid sequence shown in SEQ ID NO: 69.

In another aspect of the invention, there is provided a method of treating a human suffering from lupus nephritis (LN), comprising administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to suppress MASP-2 dependent complement activation. In one embodiment, the subject is suffering from steroid-dependent LN. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount effective to improve renal function. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion from baseline 24-hour protein excretion in the urine of the subject prior to treatment. In one embodiment, the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in the specified subject. 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 shown in SEQ ID NO: 67 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 amino acid sequence shown in SEQ ID NO: 69.

In another aspect of the invention, there is provided a method for reducing proteinuria in a human suffering from IgAN, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 the amino acid sequence shown in SEQ ID NO: 67 and the light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69 according to the following dosing regimens:

a) administering about 4 mg/kg (ie, 3.6 to 4.4 mg/kg) of said antibody to an IgAN-suffering subject intravenously once a week for a treatment period of at least 12 weeks;

b) administering about 180 mg to about 725 mg (i.e., 162 to 797 mg) of said antibody to an IgAN-suffering subject intravenously once a week for a treatment period of at least 12 weeks, wherein the method reduces proteinuria in specified subject.

Description of drawings

The foregoing aspects and many of the accompanying advantages of this invention will become more apparent with reference to the following detailed description with reference to the accompanying drawings.

Fig. 1 is a diagram illustrating the genomic structure of human MASP-2.

Fig. 2A is a schematic diagram illustrating the domain structure of the human MASP-2 protein.

Fig. 2B is a schematic diagram illustrating the domain structure of the human MAp19 protein.

Fig. 3 is a diagram illustrating the mouse MASP-2 knockout strategy.

Fig. 4 is a diagram illustrating the construction of the MASP-2 minigene.

In FIG. 5A presents results demonstrating that MASP-2 deficiency leads to loss of C4-mediated activation of the lectin pathway, as defined by the absence of C4b deposits on mannan, as described in Example 2.

In FIG. 5B presents results demonstrating that MASP-2 deficiency leads to loss of C4-mediated activation of the lectin pathway, as defined by the absence of C4b deposits on zymosan, as described in Example 2.

In FIG. 5C shows results showing the relative levels of C4 activation in serum samples derived from MASP-2+/-, MASP-2-/- lines and wild-type lines, as determined by C4b deposition on mannan and zymosan as described in Example 2.

- 8 043102

In FIG. 6 presents results demonstrating that the addition of mouse recombinant MASP-2 to MASP2-/-serum samples restored C4-mediated activation of the lectin pathway, which is protein concentration dependent, as determined by the deposition of C4b on mannan as described in Example 2.

In FIG. 7 presents the results demonstrating that the classical pathway is functional in the MASP-2-/- lineage as described in Example 8.

In FIG. 8A shows results demonstrating that anti-MASP-2 antibody Fab2 #11 inhibits the formation of C3 convertase as described in Example 10.

In FIG. 8B shows results demonstrating that anti-MASP-2 antibody Fab2 #11 binds to native rat MASP-2 as described in Example 10.

In FIG. 8C shows results demonstrating that anti-MASP-2 antibody Fab2 #41 suppresses C4 cleavage as described in Example 10.

In FIG. 9 presents the results demonstrating that all Fab2 anti-MASP-2 antibodies tested that inhibited the formation of C3 convertase also inhibited C4 cleavage as described in Example 10.

Fig. 10 is a diagram illustrating recombinant rat MASP-2 derived polypeptides that were used to map epitopes of MASP-2 blocking Fab2 antibodies as described in Example 11.

In FIG. 11 shows results showing the binding of anti-MASP-2 Fab2 nos. 40 and 60 to rat MASP-2 polypeptides as described in Example 11.

In FIG. 12A is a graphical representation of the level of MAC deposition in the presence or absence of human monoclonal anti-MASP-2 antibody (OMS646) under lectin pathway specific test conditions, indicating that OMS646 inhibits lectin-mediated MAC deposition with an IC ₅₀ value of about 1 nM as described in example 12.

In FIG. 12B is a graphical representation of the level of MAC deposition in the presence or absence of human anti-MASP-2 monoclonal antibody (OMS646) under classical pathway specific testing conditions, indicating that OMS646 does not suppress classically mediated MAC deposition as described in the example. 12.

In FIG. 12C is a graphical representation of the level of MAC deposition in the presence or absence of human anti-MASP-2 monoclonal antibody (OMS646) under alternative pathway specific testing conditions, indicating that OMS646 does not inhibit alternative-mediated MAC deposition as described in Example. 12.

In FIG. 13 is a graphical plot of the pharmacokinetic (PK) profile of a human anti-MASP-2 monoclonal antibody (OMS646) in mice showing the concentration of OMS646 (mean for n=3 animals/group) as a function of time after the indicated dose as described in Example 12 .

In FIG. 14A is a graphical representation of the pharmacodynamic (PD) response of a human monoclonal anti-MASP-2 antibody (OMS646) as measured by a drop in activity of the lectin pathway of the complement system in mice following intravenous administration as described in Example 12.

In FIG. 14B is a graphical representation of the pharmacodynamic (PD) response of the human monoclonal antibody MASP-2 (OMS646) as measured by the reduction in activity of the lectin pathway of the complement system in mice following subcutaneous administration as described in Example 12.

In FIG. 15 graphically shows the results of computer analysis of images of kidney tissue sections stained with Sirius red; tissue sections were obtained from wild-type and MASP-2-/- mice 7 days after unilateral ureteral obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice as described in Example 14.

In FIG. 16 graphically shows the results of computer analysis of images of kidney tissue sections stained with antibodies to macrophage marker F4/80; tissue sections were obtained from wild-type and MASP-2-/- mice 7 days after unilateral ureteral obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice as described in Example 14.

In FIG. 17 graphically shows the relative expression levels of collagen-4 mRNA measured by quantitative PCR (qPCR) in kidney tissue sections obtained from wild-type and MASP-2-/ mice 7 days after unilateral ureteral obstruction (UUO) and sham-operated wild-type mice and MASP-2 -/- mice as described in Example 14.

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

In FIG. 19 graphically shows the relative levels of interleukin-6 (IL-6) mRNA expression measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 −/− mice 7 days after unilateral ureteral obstruction (UUO) and sham-operated wild-type mice. type and MASP-2 -/- mice as described in Example 14.

- 9 043102

In FIG. 20 graphically shows the relative levels of interferon-γ mRNA expression measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 -/- mice 7 days after unilateral ureteral obstruction (UUO) and sham-operated wild-type mice and

MASP-2 -/- mice as described in Example 14.

In FIG. 21 graphically shows the results of computer analysis of images of kidney tissue sections stained with Sirius red; tissue sections were obtained 7 days after unilateral ureteral obstruction (UUO) in wild type mice treated with MASP-2 inhibitory antibody and isotype control antibody as described in Example 15.

In FIG. 22 graphically shows the hydroxyproline content in kidneys obtained 7 days after unilateral ureteral obstruction (UUO) in wild-type mice treated with an inhibitory MASP-2 antibody, compared with the level of hydroxyproline in occluded kidney tissue obtained from wild-type mice treated with isotype control. , IgG4 as described in example 15.

In FIG. 23 is a graphical representation of total serum proteins (mg/mL) measured on day 15 of protein overdose effects study in WT mice (n=2) treated with saline only, WT mice treated with BSA (n=6) , and MASP-2 -/- mice treated with BSA (n=6) as described in example 16.

In FIG. 24 is a graphical representation of the total amount of protein (mg) excreted in urine collected over a 24-hour period on day 15 of studying the effect of protein overdose in saline-only control WT mice (n=2), WT mice. BSA-treated (n=6) and BSA-treated MASP-2-/- mice (n=6) as described in Example 16.

In FIG. 25 shows representative sections of kidney tissue stained with hematoxylin and eosin (H&E) at day 15 of studying protein overdose rates in the following groups of mice: (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.

In FIG. 26 graphically shows the results of computer analysis of images of kidney tissue sections stained with an antibody to macrophage marker F4/80, showing the average stained area of macrophages (%), where tissue sections were obtained on the 15th day of studying the effect of protein overdose in wild-type control mice (n= 2), wild type mice treated with BSA (n=6) and MASP-2-/- mice treated with BSA (n=5) as described in Example 16.

In FIG. 27A graphically shows an analysis of the presence of a correlation between macrophage levels and proteinuria in each BSA-treated wild-type mouse (n=6) as a plot of total protein excreted measured in a 24-hour urine sample versus macrophage infiltration (mean stained area, %), as described in example 16.

In FIG. 27B graphically shows an analysis of the presence of a correlation between macrophage levels and proteinuria in each BSA-treated MASP-2-/- mice (n=5) as a plot of total protein excreted measured in a 24-hour urine sample versus macrophage infiltration ( average painted area, %), as described in example 16.

In FIG. 28 graphically shows the results of computer analysis of images of tissue sections stained with anti-TGFe antibody (measured as area of anti-TGFe antibody stained, expressed as a percentage) in wild-type mice treated with BSA (n=4) and MASP-2 -/- mice treated with BSA (n=5) as described in Example 16.

In FIG. 29 graphically shows the results of computer analysis of images of tissue sections stained with anti-TNFa antibody (measured as area of anti-TNFa antibody stained) in wild-type mice treated with BSA (n=4) and MASP-2-/- mice treated with BSA ( n=5) as described in example 16.

In FIG. 30 is a graphical representation of computer image analysis results of tissue sites stained with anti-Wh-6 antibody (measured as area stained with anti-Wh-6 antibody) 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.

In FIG. 31 graphically shows the frequency of TUNEL-positive apoptotic cells counted in sequentially selected 20 high magnification fields (HPFs) in renal cortical tissue sections obtained from WT (n=1) control, MASP-2-/- control mice. (n=1), wild-type mice treated with BSA (n=6), and MASP-2 -/- mice treated with BSA (n=7) as described in example 16.

In FIG. 32 shows representative tissue sections stained with H&E obtained on day 15 after BSA treatment of the following groups of mice: (panel A) wild-type control mice, mice treated with saline; (panel B) mice treated with isotype control antibody and (panel C) wild-type mice treated with MASP-2 inhibitory antibody as described in Example 17.

In FIG. 33 graphically shows the frequency of TUNEL-positive apoptotic cells counted in sequentially selected 20 high magnification fields of view (HPF) in tissue sections.

- 10 043102 renal cortex obtained from wild-type control mice treated with saline control and BSA (n=8), wild-type mice treated with isotype control antibody and BSA (n=8), and wild-type mice treated with inhibitory MASP -2 antibody and BSA (n=7) as described in Example 17.

In FIG. 34 is a graphical representation of computer image analysis of anti-TGFβ antibody-stained tissue areas (measured as area of anti-TGFe antibodies stained, %) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8) as described in Example 17.

In FIG. 35 graphically shows the results of computer analysis of images of tissue sections stained with TNFa antibody (measured as area of anti-TNFα antibodies stained, %) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8) as described in Example 17.

In FIG. 36 graphically shows the results of computer analysis of images of tissue sections stained with anti-IL-6 antibody (measured as area of anti-II-6 stained antibodies, %) in wild-type mice treated with BSA and saline (n=8), mice, treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8) as described in Example 17.

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

In FIG. 38 graphically shows the results of computer analysis of images of kidney tissue sections stained with F4/80 macrophage marker antibody showing the mean macrophage area stained (%) on day 14 after treatment with adriamycin or saline alone (wild-type control mice) for the following groups of mice : wild-type control mice treated with saline only; wild-type mice treated with adriamycin; MASP-2-/- mice treated with saline only and MASP-2-/- mice treated with adriamycin, where **p=0.007 as described in Example 18.

In FIG. 39 graphically shows the results of computer analysis of images of Sirius red stained kidney tissue sections showing stained areas of collagen deposition (%) at day 14 after treatment with adriamycin or saline alone (wild-type control mice) for the following groups of mice: wild-type control mice who received saline only; wild-type mice treated with adriamycin; MASP-2-/- mice treated with saline alone and MASP-2-/- mice treated with adriamycin, where **p=0.005 as described in Example 18.

In FIG. 40 is a graphical representation of the urinary albumin/creatinine ratio (uACR) in two IgA patients during a 12 week study with weekly treatment with an inhibitory MASP-2 antibody (OMS646) as described in Example 19.

In FIG. 41 shows a plot of uACR (mg/g) versus time for four IgAN patients treated with OMS646 from baseline to day 120 as described in Example 21.

In FIG. 42 graphically shows the change in protein level in the post-treatment 24 hour urine collection from baseline on pre-treatment day 1 for four IgAN patients treated with OMS646 as described in Example 21.

In FIG. 43 graphically shows the mean change in protein level in the 24-hour post-treatment urine collection from baseline pre-treatment levels for four IgAN patients treated with OMS646 as described in Example 21.

Description of the sequence list

SEQ ID NO: 1 - Human MAP19 cDNA.

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

SEQ ID NO: 3 - human MAp19 protein (mature).

SEQ ID NO: 4 - human MASP-2 cDNA.

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

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

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

Antigens: (relative to the mature MASP-2 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).

- 11 043102

- EGF region (aa 122-166).

- serine protease domain (aa 429-671).

- inactive serine protease domain (aa 610-625 with Ser618 replaced by Ala).

- TPLGPKWPEPVFGRL (CUBI peptide).

- TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide).

- TFRSDYSN (central part of MBL binding site).

- FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding site).

- IDECQVAPG (EGF peptide).

- ANMLCAGLESGGKDSCRGDSGGALV (central part that binds serino-

SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO protease).

Detailed description.

peptide inhibitors.

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

- full length MBL cDNA.

- full-length protein MBL.

- OGK-X-GP (binding consensus).

- OGKLG.

- GLRGLQGPOGKLGPOG.

- GPOGPOGLRGLQGPOGKLGPOGPOGPO.

- GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG.

- GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin).

SEQ ID NO: 28 - GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficolin p35).

SEQ ID NO: 29 - LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site).

expression inhibitors.

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

SEQ ID NO: 31 - 5' CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3', nucleotides 12-45 of SEQ ID NO: 4, including the MASP-2 translation start site (sense).

SEQ ID NO: 32 - 5' GACATTACCTTCCGCTCCGACTCCAACGAGAAG 3', nucleotides 361-396 of SEQ ID NO: 4 encoding the region containing the MASP-2 MBL binding site (sense).

SEQ ID NO: 33 - 5' AGCAGCCCTGAATACCCACGGCCGTATCCCAAA 3', nucleotides 610-642 of SEQ ID NO: 4 encoding the region containing the CUBII domain.

coding primers.

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

- CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for CUB).

- GGAATTCCTAGGCTGCATA (3' PCR for CUB).

- GGAATTCCTACAGGGCGCT (3' PCR for CUBIEGF).

- GGAATTCCTAGTAGTGGAT (3' PCR for CUBIEGFCUBII).

38-47 - cloning primers for the humanized antibody.

- a peptide bond of 9 amino acids.

Expression vector.

SEQ ID NO: 49 is the MASP-2 minigene insert.

SEQ ID NO: 50 is the mouse MASP-2 cDNA.

SEQ ID NO: 51 is the mouse MASP-2 protein (leader).

SEQ ID NO: 52 is the mature mouse MASP-2 protein.

SEQ ID NO: 53 is the rat MASP-2 cDNA.

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

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

SEQ ID NO: 56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2, which are used to obtain human MASP-2A.

SEQ ID NO: 60-63 are mouse MASP-2 site-directed mutagenesis oligonucleotides that are used to generate mouse MASP-2A.

SEQ ID NO: 64-65 are oligonucleotides for site-directed mutagenesis of rat MASP-2, which are used to obtain rat MASP-2A.

SEQ ID NO: 66 is DNA encoding the heavy chain variable region (VH) 17D20_dc35VH21N11VL (OMS646) (no signal peptide).

SEQ ID NO: 67 is the heavy chain variable region (VH) polypeptide 17D20_dc35VH21N11VL (OMS646).

SEQ ID NO: 68 is the 17N16mc heavy chain variable region (VH) polypeptide.

SEQ ID NO: 70 is DNA encoding the light chain variable region (VL) 17D20_dc35VH21N11VL (OMS646).

SEQ ID NO: 69 is the light chain variable region (VL) polypeptide 17D20_dc35VH21N11VL (OMS646).

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

- 12 043102

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

SEQ ID NO: 73 is SGMI-2M (Short Medium Length).

SEQ ID NO: 74 is SGMI-2S (short version, short).

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

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

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

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

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

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

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

SEQ ID NO: 82 is a polynucleotide encoding a polypeptide containing VH-M2ab6-SGMI-2-N and a human IgG4 constant region with a hinge mutation.

SEQ ID NO: 83 is a polynucleotide encoding a polypeptide containing VH-M2ab6-SGMI-2-C and a human IgG4 constant region with a hinge mutation.

SEQ ID NO: 84 shows a polynucleotide encoding a polypeptide containing VL-M2ab6SGMI-2-N and human Ig lambda chain constant region.

SEQ ID NO: 85 shows a polynucleotide encoding a polypeptide containing VL-M2ab6SGMI-2-C and human Ig lambda chain constant region.

Detailed description of the invention

The present invention is based on the unexpected discovery of the present inventors that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-2), a key regulator of the lectin pathway of the complement system, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including the unilateral ureteral obstruction model. (UUO), protein overdose model, and kidney fibrosis model in adriamycin-induced nephropathy. Thus, the inventors have demonstrated that inhibition of MASP-2-mediated activation of the lectin pathway provides an effective therapeutic approach to alleviate, treat, or prevent renal fibrosis, such as tubulointerstitial inflammation and fibrosis, regardless of the underlying cause. The present description also shows that the use of a MASP-2 inhibitory antibody (OMS646) effectively improves kidney function and reduces the need for corticosteroids in humans suffering from immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN).

I. Definitions.

Unless specifically stated otherwise, all terms used in the present description have the same meaning, which is clear to a person skilled in the field to which the present invention relates. The following definitions are intended to clarify the terms used in the description and in the claims of the present invention.

As used herein, the term MASP-2-dependent complement activation refers to MASP-2-dependent activation of the lectin pathway that occurs under physiological conditions (i.e., in the presence of Ca~) and that results in the formation of the lectin pathway C3 convertase C4b2a and accumulation of a C3 cleavage product, such as C3b, and then the formation of C5-convertase C4b2a(C3b)n, which was determined to be the main cause of opsonization.

As used herein, the term alternative pathway refers to complement activation that is initiated, for example, by zymosan from fungal and yeast cell walls, gram-negative bacteria outer membrane lipopolysaccharide (LPS), and rabbit erythrocytes, as well as a variety of pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which, as is commonly believed, arises as a result of spontaneous proteolytic cleavage of complement factor C3 with the formation of C3b.

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

As used herein, the term classical pathway refers to complement activation that is triggered by an antibody bound to a foreign particle and that requires binding to a C1q recognition molecule.

As used herein, the term MASP-2 inhibitory agent means any agent that binds or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, including anti-MASP-2 antibodies and their MASP-2 binding fragments, natural and synthetic peptides, small molecules, soluble MASP-2 receptors,

- 13 043102 expression inhibitors and isolated natural inhibitors; as well as peptides that, upon activation of the lectin pathway, compete with MASP-2 for binding to other recognition molecules (for example, MBL, H-ficolin, M-ficolin, or L-ficolin), while this term does not cover antibodies that bind to other recognition molecules. The MASP-2 inhibitory agents used in the present invention can reduce the level of MASP-2-mediated complement activation by more than 20%, eg more than 50%, eg more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces the level of MASP-2-mediated complement activation by more than 90% (ie, the level of MASP-2-mediated complement activation will result in only 10% or less).

As used herein, the term fibrosis refers to the formation or presence of excess connective tissue on 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 excess extracellular matrix. The normal physiological response to injury results in the formation of connective tissue as part of the healing process, but this formation of connective tissue can be delayed and become pathological, altering tissue architecture and function. 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 treatment of fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused by or complicated by fibrosis and/or inflammation means arresting, attenuating, alleviating or suppressing fibrosis in said mammal.

As used herein, the term proteinuria means the presence of protein in human urine in an abnormal amount, for example, in excess of 0.3 g of protein in human urine collected in 24 hours, or in a concentration in excess of 1 g/L. In some embodiments, a subject suffering from proteinuria refers to a subject, such as a subject suffering from immunoglobulin A (IgA) nephropathy, whose urinary protein amount exceeds 1.0 g of protein in a 24-hour urine collection.

As used herein, the term improvement in proteinuria or reduction in proteinuria means a reduction in a subject suffering from proteinuria of 24-hour urinary protein excretion of at least 20%, such as at least 30%, such as at least 40%, such as by at least 50% or more compared to the subject's baseline 24 hour urinary protein excretion prior to treatment with the MASP-2 inhibitory agent. In one embodiment, treatment with a MASP-2 inhibitory agent according to the method of the invention is effective in reducing proteinuria in a human such that a greater than 20% reduction in 24-hour urinary protein excretion is achieved, or, for example, a greater than 30% reduction in 24- hourly urinary protein excretion, or, for example, a greater than 40% reduction in 24-hour urinary protein excretion, or, for example, a greater than 50% reduction in 24-hour urinary protein excretion.

As used herein, the term antibody encompasses antibodies and fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including humans) or obtained by hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods). obtaining antibodies or antibody fragments), wherein said antibodies or fragments thereof specifically bind to a target polypeptide, such as, for example, MASP-2 polypeptides or regions thereof. This does not mean that the term antibody is limited to the sources of origin of antibodies specified in the present description or methods for their production (for example, using hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multivalent, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric antibodies; monoclonal antibodies mouse-human, mouse-primate, primate-human; and anti-idiotypic antibodies, which may be intact antibodies, or fragments thereof. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab')2, Fv), single chain fragments (ScFv), their synthetic variants, natural variants, fusion proteins comprising part of an antibody together with an antigen-binding fragment with the desired specificity, humanized antibodies, chimeric antibodies, and any modified configuration of immunoglobulin molecules containing an antigen-binding site or fragment (epitorecognition site) with the desired specificity.

Monoclonal antibody means a homogeneous population of antibodies in which the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring) that participate in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term monoclonal antibody covers not only

- 14 043102 intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab') ₂ , Fv), single-chain fragments (ScFv), variants thereof, recombinant proteins containing antigen-binding part, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope-recognition site) with the desired specificity and ability to bind to an epitope. This does not mean that this term is limited to the sources of origin of antibodies indicated in the present description or methods for their production (for example, using hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term includes whole immunoglobulins, as well as fragments, etc., described above under the definition of antibody.

As used herein, the term antibody fragment refers to a portion derived from or related to a full-length antibody, such as, for example, an anti-MASP-2 antibody, typically comprising the antigen-binding or variable region of that antibody. Representative 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 contains the VH and VL domains of the antibody, and these domains are present in the same polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, which allows the scFv to form the desired structure for antigen binding.

As used herein, the term chimeric antibody is a recombinant protein that contains variable domains and complementarity determining regions derived from a non-human (eg, rodent) antibody, while the remainder of the antibody molecule is derived from a human antibody.

As used herein, the term humanized antibody is a chimeric antibody containing a minimum sequence that corresponds to specific complementarity determining regions derived from a non-human immunoglobulin transplanted into a human antibody scaffold. 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 Mannan Binding Protein (MBP).

As used herein, the term membrane attack complex (MAC) means a complex of five terminal complement components (C5b combined with C6, C7, C8 and C9) that inserts into and destroys membranes (also referred to as C5b-9).

As used herein, the term subject includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

The amino acid residues used herein 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 ).

In a broader sense, natural amino acids can be divided into groups based on the chemical properties of the side chains of the respective amino acids. Hydrophobic amino acids are Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. Hydrophilic amino acids are Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This group of amino acids can also be divided into subclasses. The uncharged hydrophilic amino acids are Ser, Thr, Asn or Gln. The acidic amino acids are Glu or Asp. The main amino acids are Lys, Arg or His.

As used herein, the term conservative amino acid substitution is represented by amino acid substitutions within one of the following groups:

(1) glycine, alanine, valine, leucine and isoleucine;

(2) phenylalanine, tyrosine and tryptophan;

(3) serine and threonine;

(4) aspartate and glutamate;

(5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

As used herein, the term oligonucleotide means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term also covers those oligonucleobases that are composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) bonds, as well as oligonucleotides that do not occur in

- 15 043102 the nature of the modification.

As used herein, the term epitope refers to a region on a protein (e.g., protein

MASP-2) to which the antibody binds. Overlapping epitopes include at least one (eg, two, three, four, five or six) common amino acid residue(s), including linear and non-linear epitopes.

As used herein, the terms polypeptide, peptide, and protein are used interchangeably and refer to any amino acid chain associated with a peptide, regardless of length or post-translational modification. The MASP-2 protein disclosed herein may contain or be a wild-type protein, or may be a variant that has 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 one of the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, a human MASP-2 protein may have an amino acid sequence that is 70% or more identical (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%) a human MASP-2 protein having the amino acid sequence shown in SEQ ID NO: 5.

In some embodiments, the peptide fragments 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, 400, 450, 500, or 600 or more) amino acid residues (eg, at least 6 contiguous amino acid residues from SEQ ID NO: 5). In some embodiments, the human MASP-2 antigenic peptide fragment 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, 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 (eg, less than 500 contiguous amino acid residues anywhere in SEQ ID NO: 5).

Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a control sequence after sequence alignment and gaps have been introduced, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent sequence identity can be achieved in various ways that are within the skill of the art, for example, using open source software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences, can be determined by known methods.

Used in this application, the term approximately or approximately in front of a numerical value indicates this value plus or minus 10% of the specified value.

II. Brief description of the invention.

As disclosed herein, the inventors have identified the central role of the lectin pathway in the initiation and progression of renal tubular disease, implying a key role for lectin pathway activation in the pathophysiology of a wide range of renal diseases, including IgA nephropathy, C3 glomerulopathy, and other glomerulonephritis. The present description also states that the present inventors have discovered that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-2), a key regulator of the lectin pathway of the complement system, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including the unilateral ureteral obstruction model. (UUO), protein overdose model, and kidney fibrosis model in adriamycin-induced nephropathy. Thus, the inventors have shown that the suppression of MASP-2-mediated activation of the lectin pathway provides an effective therapeutic approach for the alleviation, treatment or prevention of renal fibrosis, such as tubulointerstitial fibrosis, regardless of the underlying cause.

Lectins (MBL, M-fcolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that trigger the complement system through the mechanism of innate immunity, and such a system includes the lectin initiation pathway and its associated terminal pathway amplification loop. , which enhances lectin-initiated activation of terminal effector complement molecules. C1q is a specific recognition molecule that triggers the complement system through the mechanism of acquired immunity, and such a system includes the classical initiation pathway and its associated terminal pathway enhancement loop that enhances C1q-initiated activation of terminal complement effector molecules. The authors refer to these two major complement activation systems, such as lectin-dependent

- 16 043102 complement system and Clq-dependent complement system, respectively.

In addition to its important role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent the development of these side effects. Establishing the possibility of suppressing the MASP-2-mediated lectin pathway, while not affecting the classical pathway, provides an opportunity for highly desirable specific suppression of the activation of only the complement system that causes a particular pathology, without completely suppressing the ability of complement to immune defense. Thus, for example, in pathological conditions in which complement activation is mediated predominantly by the lectin-dependent complement system, specific suppression of this system alone is preferable. To regulate the processing of the immune complex and to protect the host from infection, the system of C1q-dependent complement activation must remain intact.

The preferred target protein component for the development of therapeutic agents for the specific suppression of the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D, and properdin), only MASP-2 is unique and essential for function. this lectin-dependent complement system. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components of the lectin-dependent complement system. However, the loss of any one of these lectin components will not necessarily suppress the activation of this system, which is due to the excess amount of lectin. To ensure suppression of the activation of the lectin-dependent complement system, all five lectins must be inhibited. Furthermore, since MBL and ficolins are also known to have complement-independent opsonin activity, suppression of lectin function would result in the loss of this valuable host defense mechanism against infection. In contrast, this complement-independent lectin opsonin activity will remain intact if MASP-2 is the target of inhibition. Another advantage of MASP-2 as a therapeutic target for suppressing the activation of the lectin-dependent complement system is that the plasma concentration of MASP-2 is the lowest (~500 ng/mL) of any other complement protein, and therefore for complete suppression will be sufficient at low concentrations of high affinity MASP-2 inhibitors (Moller-Kristensen, M. et al., J. Immunol. Methods, 282:159-167, 2003).

As described in Example 14 of the present disclosure, in an animal model of fibrotic kidney disease (UUO unilateral ureteral obstruction), it was determined that kidney disease in mice lacking the MASP-2 gene (MASP-2-/-) manifested to a significantly lesser extent compared to with wild type controls as evidenced by inflammatory cellular infiltrates (75% reduction) and histological markers of fibrosis such as collagen deposition (one third reduction). Example 15 further demonstrates that wild-type mice administered systemically with a monoclonal anti-MASP-2 antibody that selectively blocks the lectin pathway while leaving the classical pathway intact were protected from kidney fibrosis compared to wild-type mice administered with an isotype control antibody. These results indicate that the lectin pathway is a key factor in causing kidney disease and further demonstrate that a lectin pathway blocking MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective as an anti-fibrotic agent. Example 16 additionally showed in a protein overdose model that wild-type mice that received bovine serum albumin (BSA) developed proteinuric nephropathy, while MASP-2-/- mice that received the same dose of BSA developed kidney damage in lesser degree. Example 17 shows that wild-type mice treated systemically with a monoclonal anti-MASP-2 antibody that selectively blocks the lectin pathway while leaving the classical pathway intact were protected from kidney injury in a protein overdose model. Example 18 shows that MASP-2 −/− mice exhibited renal inflammation and tubulointerstitial damage in an adriamycin-induced nephrological model of renal fibrosis to a lesser extent compared to wild-type mice. Example 19 shows that in an ongoing phase 2 open-label clinical study in renal disease, patients with IgA nephropathy who were administered an anti-MASP-2 antibody had a clinically significant and statistically significant decrease in the urinary albumin/creatinine ratio at the end of treatment ( uACR) throughout the study, as well as a decrease in the level of protein in the urine collected within 24 hours, compared with baseline. Example 19 also shows that in the same phase 2 study, patients with membranous nephropathy treated with an anti-MASP-2 antibody also experienced a decrease in uACR during treatment. Example 20 shows that in an ongoing phase 2 open-label kidney study at the end of treatment, 4 out of 5 patients with lupus nephritis (LN) treated with an anti-MASP-2 antibody had a clinically significant decrease in protein levels at 24-hour urine collection relative to baseline.

In accordance with the foregoing, the present invention relates to the use of inhibitory

- 17 043102

MASP-2 agents, such as MASP-2 inhibitory antibodies, as anti-fibrotic agents, to the use of MASP-2 inhibitory agents for the manufacture of a medicament for the treatment of a fibrotic condition, and to methods for preventing, treating, alleviating, or relieving a fibrotic condition in a person in need thereof wherein said method comprises administering to said patient an effective amount of a MASP-2 inhibitory agent (eg, an anti-MASP-2 antibody).

The methods of the invention may be used to prevent, treat, alleviate, or reverse a fibrotic condition in a human suffering from any disease or disorder caused or complicated by fibrosis and/or inflammation, including kidney disease (e.g., chronic kidney disease, IgA nephropathy, C3- glomerulopathy and other glomerulonephritis), lungs (eg, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (eg, cirrhosis, nonalcoholic fatty liver disease), heart (eg, myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis), brain (eg, stroke), skin (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids), vascular system (eg, atherosclerotic vascular disease), bowel (eg, Crohn's disease), eyes (eg, anterior subcapsular cataract, opacification of the posterior capsules), soft tissue structures of the musculoskeletal system (eg, cellular capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (eg, endometriosis, Peyronie's disease), and some infectious diseases (eg, alphavirus, hepatitis C and hepatitis B).

III. The role of MASP-2 in diseases and conditions caused or complicated by fibrosis.

Fibrosis is the formation or presence of excess connective tissue in an organ or tissue, usually in response to injury or injury. The hallmark of fibrosis is the production of excess extracellular matrix after injury. In the kidney, fibrosis is characterized as a progressive detrimental deposition of connective tissue on the renal parenchyma, which inevitably leads to a decrease in kidney function, regardless of the primary kidney disease that causes the initial damage. The so-called epithelial-mesenchymal transition (EMT), a change in cellular characteristics in which tubular epithelial cells transform into mesenchymal fibroblasts, constitutes the main mechanism of renal fibrosis. Fibrosis affects almost all tissues and organ systems and can occur as a repair or replacement response to a stimulus such as tissue injury or inflammation. The normal physiological response to injury leads to accumulation of connective tissue, but if this process becomes pathological, replacement of highly differentiated cells by scarring of the connective tissue alters tissue structure and function. 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 currently no effective treatment or therapy for fibrosis, but animal studies and anecdotal reports of human treatment suggest that fibrotic tissue lesions can be reversed (Tampe and Zeisberg, Nat. Rev. Nephrol., vol 10:226 -237, 2014).

Many diseases result from fibrosis that causes progressive organ failure, including kidney disease (eg, chronic kidney disease, IgA nephropathy, C3 glomerulopathy, and other glomerulonephritis), lung (eg, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver ( eg, cirrhosis, non-alcoholic fatty liver disease), heart (eg, myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis), brain (eg, stroke), skin (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids), vascular system (eg, atherosclerotic vascular disease), bowel (eg, Crohn's disease), eyes (eg, anterior subcapsular cataract, posterior capsular opacity), soft tissue structures of the musculoskeletal system (eg, cellular capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (eg endometriosis, Peyronie's disease) and certain infectious diseases (eg alpha virus, hepatitis C and hepatitis B, etc.).

Although fibrosis occurs in many tissues and in many diseases, there are common molecular and cellular mechanisms in its pathology. The deposition of extracellular matrix by fibroblasts is accompanied by infiltrates of immune cells, predominantly mononuclear cells (see Wynn T., Nat. Rev. Immunol., 4(8):583-594, 2004, incorporated herein by reference). An active inflammatory response leads to 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), degradation enzymes (elastase , matrix metalloproteinases, cathepsins) and extracellular matrix proteins (collagens, fibronectin, integrins).

In addition, the complement system becomes active in numerous fibrotic diseases. Complement components, including the membrane attack complex, have been identified in numerous fibrous tissue samples. For example, components of the lectin pathway have been found in fibrotic lesions of the kidneys (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 diseases (Rensen et al., Hepatology, 50(6):1809-17 (2009)); and lung diseases (Olesen et al., Clin. Immunol., 121(3):324-31 (2006)).

- 18 043102

It has been found that excessive activation of the complement system is one of the key factors in the development of immune complex-mediated, as well as antibody-independent, glomerulonephritis. However, there is strong evidence that uncontrolled in situ complement activation is associated with 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; Mathern, D.R. et al., Clin. J. Am. Soc. Nephrol., 10:P1636-1650, 2015). Strong pro-inflammatory signals caused by local complement activation may be initiated by complement components filtered into the proximal tubule and subsequently into the interstitial space, or by abnormal synthesis of complement components by tubular or other resident and infiltrating cells, or by altered expression of complement regulatory proteins in kidney cells, or absence or loss or increase of functional mutations in complement regulatory components (Mathern D.R. et al., Clin. J. Am. Soc. Nephrol., 10:P1636-1650, 2015, Sheerin N.S. et al., FASEB J., 22 :1065-1072, 2008). For example, in mice, deficiency of the regulatory protein/gene (Crry) associated with the complement regulatory protein CR1 results in complement activation in a tubulointerstitial (TI) lesion pattern, followed by inflammation and fibrosis characteristic of the lesion seen in human TI diseases (Naik A. et al., Semin. Nephrol., 33:575-585, 2013, Bao L. et al., J. Am. Soc. Nephrol., 18:811-822, 2007). Exposure of tubular epithelial cells to anaphylatoxin C3a leads to an epithelial-mesenchymal transition (Tsang Z. et al., J. Am. Soc. Nephrol., 20:593-603, 2009). It has recently been shown that blocking C3a signaling through the C3a receptor alone reduces TI renal fibrosis in animals with proteinuric and nonproteinuric diseases (Tsang Z. et al., J. Am. Soc. Nephrol., 20:593-603, 2009; Bao L. et al., Kidney Int., 80:524-534, 2011).

As disclosed herein, the inventors have identified a central role for the lectin pathway in the initiation and progression of renal tubular pathology, implying a key role for lectin pathway activation in the pathophysiology of a wide range of kidney diseases, including IgA nephropathy, C3 glomerulopathy, and other glomerulonephritis (Endo M. et al., Nephrol Dialysis Transplant., 13:1984-1990, 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., Transplant. Nephrol. Disysis ., 14:881-886, 1999; Pickering et al., Kidney International, 84:1079-1089, 2013), diabetic nephropathy (Hovind P. et al., Diabetes, 54:1523-1527, 2005), ischemic reperfusion lesion (Asgari E. et al., FASEB J., 28:3996-4003, 2014) and transplant rejection (Berger S. P. et al., Am. J. Transplant., 5:1361-1366, 2005).

As further shown herein, the inventors have demonstrated that downregulation of MASP-2 reduces inflammation and fibrosis in mouse models of tubulointerstitial disease. Therefore, MASP-2 inhibitory agents are expected to be useful in the treatment of renal fibrosis, including tubulointerstitial inflammation and fibrosis, proteinuria, IgA nephropathy, C3 glomerulopathy and other glomerulonephritis and renal ischemic reperfusion injury.

Kidney diseases and disorders.

According to the National Kidney Foundation, 26 million American adults have chronic kidney disease (CKD). Most patients have progressive disease leading to kidney failure requiring treatment with erythropoiesis-stimulating drugs, dialysis, or kidney transplantation to survive. There are several medications that can treat the main symptom of CKD, hypertension, but there are currently no medications that address its root cause.

Studies have shown that progressive kidney damage is caused by capillary hypertension in substructures of the kidney known as nephrons (Whitworth J.A., Annals. Acad. of Med., vol. 34(1):2005). As the nephrons (filtering units of the kidneys) are damaged or destroyed in this process, inflammation occurs and tissue scarring occurs, resulting in the replacement of nephrons with non-functional scar tissue. As a result, the ability of the kidney to filter blood decreases over time. This is called kidney fibrosis, which is a common process of progressive kidney disease. Regardless of the nature of the initial lesion, renal fibrosis is considered the common end process by which kidney disease progresses to end-stage renal disease.

Attenuation of renal fibrosis can be determined by one or more of the following: interstitial volume scores, collagen IV deposition, and/or levels of mRNA containing genes that control connective tissue growth. The compounds and methods described herein are useful in the treatment of renal fibrosis.

Fibrosis and inflammation of the kidneys are characteristic features of advanced kidney disease of almost any etiology (see Boor P. et al., J. of Am Soc. Nephrology., 18:1508-1515, 2007; and Chevalier et al., Kidney International, 75:1145-1152, 2009). Kidney failure can be caused by a heterogeneous group of disorders. Progressive kidney dysfunction leads to proteinuria and kidney failure. As the patient's health deteriorates, dialysis may be required simply to prevent kidney damage and multisystem failure. So

- 19 043102 Over time, kidney damage and kidney failure can progress to end-stage renal disease (ESRD), which is complete or near-complete, irreversible loss of kidney function. Depending on the form of kidney disease, kidney function may be lost over days or weeks, or it may slowly and gradually deteriorate over decades. Once a patient's disease has progressed to ESRD, dialysis (hemodialysis or peritoneal dialysis) is required to prevent the patient's death. Patients need to be maintained on some form of dialysis regimen or given a kidney transplant.

Components of the lectin pathway have been found in fibrotic lesions in renal 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)). In IgA nephropathy, patients with glomerular MBL deposition exhibited greater proteinuria, decreased renal function, lower serum albumin levels, more severe histology, and elevated blood pressure compared with patients without MBL deposition (Liu et al., Clin. Exp. Immunol ., (1):152-60). Increased MBL levels and lectin pathway activity.

It has also been demonstrated that C5 deficiency results in significant attenuation of major components of renal fibrosis in a non-proteinuric model of primary tubulointerstitial disease, namely unilateral ureteral obstruction (UUO) (Boor P. et al., J. of Am. Soc. of Nephrology, 18: 1508-1515, 2007). There are also reports of increased C3 gene expression in wild-type mice after UUO and a significant decrease in collagen deposits in C3-/- mice after UUO compared to wild-type mice, suggesting a role for complement activation in renal fibrosis (Fearn et al., Mol. Immunol., 48:1666-1733, 2011, abstract). However, prior to the discovery by the present inventors described herein, the complement components involved in renal fibrosis were not well defined. As described in Examples 14-17 of the present application, the present inventors unexpectedly determined that MASP-2 deficiency or blockade with an inhibitory MASP-2 antibody that selectively blocks the lectin pathway without affecting the classical pathway clearly provides protection for mice from renal fibrosis in various animals. models of kidney disease.

Accordingly, in some embodiments, a method for suppressing kidney fibrosis in a subject suffering from a kidney disease or disorder caused by or complicated by fibrosis and/or inflammation is provided, comprising administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to the subject in need thereof. The method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress renal fibrosis in a subject suffering from a kidney disease or disorder caused or complicated by fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by local application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, inhaled, nasally, subcutaneously, or by other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until the condition is controlled.

In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying kidney disease or condition. In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are administered in combination with a dialysis or plasmapheresis regimen. In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are used to reduce the frequency of dialysis or plasmapheresis required. In some other embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are used in combination with kidney transplantation. In some other embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are used to control kidney failure and prevent further deterioration of kidney function in patients awaiting kidney transplantation.

By way of example, in some embodiments, anti-MASP-2 antibodies are used to suppress renal fibrosis and thereby treat or alleviate (including treat or alleviate disease symptoms) glomerular diseases such as focal segmental glomerulosclerosis and nephrotic syndrome. Examples of symptoms that can be treated are, among others, hypertension, proteinuria, hyperlipidemia, hematuria, and hypercholesterolemia. In some embodiments, the MASP-2 inhibitory agent inhibits tubulointerstitial fibrosis. In some embodiments, the treatment includes improving kidney function, reducing proteinuria, improving hypertension, and/or reducing renal fibrosis. In some embodiments, the treatment includes

- 20 043102 (i) delaying or preventing the progression of kidney failure, the decline of kidney function or the development of ESRD;

(ii) delaying, reducing the frequency or eliminating the need for dialysis; or (iii) delaying or eliminating the need for a kidney transplant.

Some specific diseases and disorders of the kidneys caused or complicated by fibrosis and/or inflammation are described below.

In some embodiments, the kidney disease caused or complicated by fibrosis and/or inflammation is a glomerular disease, such as focal segmental glomerulosclerosis (FSGS). Glomerular diseases affect the glomeruli, allowing proteins and sometimes red blood cells to leak into the urine. Sometimes glomerular disease also prevents the kidneys from clearing waste products, so they begin to accumulate in the blood. Symptoms of glomerular disease include proteinuria, hematuria, decreased glomerular filtration rate, hypoproteinemia, and edema. Several different diseases can lead to the development of glomerular disease. This may be a direct result of an infection or drugs that are toxic to the kidneys, or it may be the result of a disease that affects the entire body, such as hypertension, diabetes, or lupus. ESRD is one of the glomerular diseases, but even this special condition, characterized by scarring in the kidneys, can have many causes. Patients with ESRD typically reach end-stage renal disease within 5-20 years, although patients with aggressive forms of the disease reach ESRD in 2-3 years.

In some embodiments, the kidney disease caused or complicated by fibrosis and/or inflammation is diabetic nephropathy (DN), which is a field of medicine requiring immediate solutions. Diabetic nephropathy is a kidney disease or kidney dysfunction that is complicated by diabetes. The condition is exacerbated by high blood pressure, high blood sugar, and high cholesterol and lipid levels. The exact cause of diabetic nephropathy is unknown. However, without wishing to be bound by a particular theory, it is believed that uncontrolled high blood sugar leads to kidney damage such as fibrosis and tissue scarring. In humans, DN manifests itself as a clinical syndrome that consists of albuminuria, a gradually decreasing glomerular filtration rate (GFR), and an increased risk of cardiovascular disease. Diabetic albuminuria is associated with the development of characteristic histopathological features, including thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix. As albuminuria and renal failure develop, glomerulosclerosis, arterioral hyalinosis, and tubulointerstitial fibrosis develop.

Accordingly, in one embodiment, the present disclosure provides methods for treating diabetic nephropathy comprising administering an effective amount of a MASP-2 inhibitory agent (eg, a MASP-2 inhibitory antibody) to a subject in need thereof. In some embodiments, the implementation of the treatment includes reducing one or more symptoms of diabetic nephropathy. In some embodiments, the implementation of the treatment includes reducing, slowing down or eliminating the need for dialysis. In some embodiments, the implementation of the treatment includes reducing, slowing down or eliminating the need for kidney transplantation. In some embodiments, the treatment includes delaying, preventing, or reversing the progression of diabetic nephropathy to renal failure or end-stage renal disease.

In some embodiments, the kidney disease caused or complicated by fibrosis and/or inflammation is lupus nephritis. As detailed below, lupus nephritis, which is a serious complication of systemic lupus erythematosus (SLE), is another example of renal fibrosis that can be treated with MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies).

Accordingly, in one embodiment, the present disclosure comprises methods of suppressing kidney fibrosis in a subject suffering from a kidney disease or disorder caused by or complicated by fibrosis and/or inflammation, comprising administering an effective amount of a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody). In some embodiments, the renal disease or disorder complicated by fibrosis and/or inflammation is selected from the group consisting of chronic kidney disease, chronic renal failure, glomerular disease (e.g., focal segmental glomerulosclerosis), immune complex disorder (e.g., IgA nonfarpathies, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial disease and C3 glomerulopathy or other types of glomerulonephritis.

Methods for preventing or treating kidney injury caused by drug-induced toxicity.

Another cause of kidney damage includes drug-induced toxicity. For example, nephrotoxins can have a direct toxic effect on tubular epithelial cells. As disclosed herein, the inventors have demonstrated that MASP-2 deficient mice are protected from adriamycin-induced nephropathy.

Nephrotoxins include, without limitation, therapeutic drugs (eg, cisplatin, gentamicin, cephaloridine, cyclosporine, amphotericin, adriamycin), radiocontrast dye, pesticides (eg, paraquat), and environmental pollutants (eg, trichlorethylene and dichloroacetylene). Other examples include puromycin aminonucleoside (PAN); aminoglycosides such as gentamicin; cephalosporins such as cephaloridine; calcineurin inhibitors such as tacrolimus or sirolimus. Drug-induced nephrotoxicity can also be caused by nonsteroidal anti-inflammatory drugs, antiretrovirals, anticytokines, immunosuppressants, cancer drugs, or ACE inhibitors. In addition, drug-induced nephrotoxicity can be caused by the abuse of nalgesin, ciprofloxacin, clopidogrel, cocaine, cox-2 inhibitors, diuretics, foscamet, gold, ifosfamide, immunoglobin, Chinese herbs, interferon, lithium, mannitol, mesalamine, mitomycin, nitrosourine other, penicillamine , penicillin, pentamidine, quinine, rifampin, streptozocin, sulfonamides, ticlopidine, triamterene, valproic acid, doxorubicin, glycerin, cidofovir, tobramycin, neomycin sulfate, colistimethate, vancomycin, amikacin, cefotaxime, cisplatin om, acyclovir, lithium, interleukin-2, cyclosporine, or indinavir.

Accordingly, in one embodiment, a subject at risk of developing or suffering from kidney damage may be receiving one or more therapeutic drugs that have a nephrotoxic effect. These subjects may be administered the MASP-2 inhibitors of the invention prior to or concurrently with such therapeutic agents. Similarly, MASP-2 inhibitors may be administered following administration of a therapeutic agent to treat or reduce the likelihood of developing nephrotoxicity.

Diseases and conditions associated with proteinuria.

It has been found that impaired glomerular filtration of protein leads to proteinuria and accelerates the progressive loss of nephrons that occurs in all chronic kidney diseases (Remuzzi and Bertani, New Eng. J. Med., vol. 339(20):1448-1456, 1998). For example, in the study described by Eddy et al., Am. J. Pathol., 135:719-33, 1989, glomerular filtration of albumin was invariably accompanied by the development of interstitial lesions and scarring. Eddy et al., 1989, also noted that complement C3 deposition on the luminal surface of the proximal tubules was observed in rats with protein overdose nephropathy, indicating that components of the complement system filtered in the glomeruli may cause interstitial damage. Complement depletion or absence of C6 has been shown to alleviate tubulointerstitial injury in proteinuric animal models such as mesangioproliferative glomerulonephritis, adriamycin nephropathy, 5/6 nephrectomy, and puromycin aminonucleoside nephrosis (Boor et al., J. of Am. Soc. of Nephrol. , JASN, 18:1508-1515, 2007). Human studies have shown that proteinuria is an independent predictor of chronic kidney disease progression and that reduction in proteinuria is renal protective (Ruggenenti P. et al., J. Am. Soc. Nephrol., 23:1917-1928, 2012).

Accordingly, in one embodiment, the present invention relates to methods for preventing or reducing proteinuria and/or preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory agent). antibodies) effective to reduce or prevent proteinuria in a subject. In one embodiment, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, exlampsia, kidney toxicity, amyloidosis, vascular collagen diseases (e.g., systemic lupus erythematosus), dehydration, glomerular diseases (e.g., membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy (eg, Berger's disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (eg, adriamycin) or opiates (e.g. heroin)); Fabry disease, infections (eg, HIV, syphilis, hepatitis A, B, or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), ebola hemorrhagic fever, nail patella syndrome, familial fever, HELLP syndrome, systemic lupus erythematosus, granulomatosis Wegener, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, Schonlein purpura - 22 043102

Genoch, urinary tract infection that has spread to the kidneys, Sjögren's syndrome and post-infectious glomerulonephritis.

Liver disease.

Liver fibrosis, also called hepatic fibrosis, is caused by the accumulation of scar tissue in the liver and is common in most types of liver disease. Replacing healthy liver tissue with scar tissue reduces the liver's ability to function properly. If the condition causing scarring is not treated, liver fibrosis can progress to cirrhosis of the liver and complete liver failure, a life-threatening condition. The main causes of liver fibrosis are alcohol abuse, chronic hepatitis C virus infection, non-alcoholic steatohepatitis, and hepatotoxicity (eg, drug-induced liver injury caused by acetaminophen or another drug).

Components of the lectin pathway have been found in fibrotic liver lesions (Rensen et al., Hepatology, 50(6):1809-17 (2009)). For example, in non-alcoholic steatohepatitis (also known as fatty liver disease), activation of complement proteins is widespread and their expression is associated with disease severity (Rensen et al., Hepatology, 50(6):1809-17 (2009), and in addition to deposits C3 and C9, accumulation of MBL was found, which confirms the activation of the lectin pathway.

Accordingly, in some embodiments, a method for suppressing hepatic fibrosis in a subject suffering from a liver disease or disorder caused by or complicated by fibrosis and/or inflammation is provided, comprising administering to the subject in need thereof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress hepatic fibrosis in a subject suffering from a liver disease or disorder caused or complicated by fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying liver disease or condition.

In some embodiments, the liver disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of cirrhosis, non-alcoholic fatty liver disease (steatohepatitis), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to to acute or chronic hepatitis, biliary disease, and liver toxicity (eg, hepatotoxicity resulting from drug-induced liver injury caused by acetaminophen or another drug).

Lung disease.

Pulmonary fibrosis is the formation or development of excess fibrous connective tissue in the lungs, with normal lung tissue being replaced by fibrous tissue. This scarring leads to lung stiffness and impaired lung structure and function. In humans, pulmonary fibrosis is thought to be the result of repeated tissue damage within and between the tiny air sacs (alveoli) in the lungs. In experimental studies, aspects of human disease have been recreated in various animal models. For example, a foreign agent such as bleomycin, fluorescein isothiocyanate, silica, or asbestos may be injected into the trachea of an animal (GharaeeKermani et al., Animal. Models. of Pulmonary Fibrosis. Methods Mol. Med., 2005, 117:251-259 ).

Accordingly, in some embodiments, a method of suppressing pulmonary fibrosis in a subject suffering from a pulmonary disease or disorder caused by or complicated by fibrosis and/or inflammation is provided, comprising administering to the subject in need thereof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody. The method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress pulmonary fibrosis, reduce pulmonary fibrosis, and/or improve lung function. Improvement in lung function symptoms includes improvement in lung function and/or capacity, reduction in fatigue, and improvement in oxygen saturation.

In some embodiments, the present invention relates to a method of treating, suppressing, preventing, or alleviating pulmonary fibrosis in a subject suffering from cystic fibrosis, comprising administering to the subject in need thereof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody.

The MASP-2 inhibitory composition may be administered locally to the area of fibrosis, eg, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, eg, via a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, eg, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying lung disease or condition.

Some specific lung diseases and disorders caused or complicated by fibrosis and/or inflammation are described below.

In some embodiments, the lung disease caused or complicated by fibrosis and/or inflammation is chronic obstructive pulmonary disease (COPD). COPD is a disease in which the walls of the airways are fibrotic with an accumulation of myofibroblasts and collagen and is the leading cause of disability and the fourth leading cause of death in the United States. In COPD, airflow is blocked and the patient's breathing becomes difficult. COPD is caused by damage to the airways that ultimately prevents the exchange of oxygen and carbon dioxide in the lungs. COPD includes chronic obstructive bronchitis and emphysema, and often both. Patients with COPD whose lungs are already damaged and whose lung function is already compromised are at increased risk for complications associated with bacterial and viral infections.

Accordingly, in one embodiment, the present invention provides methods for treating chronic obstructive pulmonary disease (COPD) comprising administering to a subject in need thereof an amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) effective to suppress and/or reduce fibrosis of the lung. In some embodiments, the implementation of the treatment includes reducing one or more symptoms of COPD. Symptoms of COPD and/or pulmonary fibrosis include, but are not limited to, cough with mucus, shortness of breath (dyspnea) that may worsen with moderate activity, fatigue, frequent respiratory infections, wheezing, chest tightness, irregular heartbeats (arrhythmia), need for a breathing machine, and oxygen therapy, right-sided heart failure or pulmonia (swelling of the heart and heart failure due to chronic lung disease), pneumonia, pneumothorax, severe weight loss, and malnutrition. Symptoms also include decreased lung function as assessed by testing using one or more standard lung function tests.

In some embodiments, the lung disease caused or complicated by fibrosis and/or inflammation is scleroderma-associated pulmonary fibrosis. As described in more detail below, scleroderma-associated pulmonary fibrosis is another example of pulmonary fibrosis that can be treated with MASP-2 inhibitors (eg, MASP-2 inhibitory antibodies).

In some embodiments, the lung disease or lung dysfunction caused or complicated by fibrosis and/or inflammation is selected from the group consisting of chronic obstructive pulmonary disease, cystic fibrosis, scleroderma-associated pulmonary fibrosis, bronchiectasis, and pulmonary hypertension.

Cardiovascular diseases.

The cause of a number of different cardiovascular pathologies is a general fibrotic process. Excessive deposition of fibrous tissue in the heart leads to cardiac pathology in which the production of extracellular matrix proteins alters the structure, architecture, shape of the heart and affects its contractile function (Khan and Sheppard, Immunology, 118:10-24, 2006).

Research suggests that fibrosis may contribute significantly to cardiac dysfunction in ischemic, dilated, and hypertrophic cardiomyopathy. For example, patients with chronic atrial fibrillation have been shown to have higher levels of interstitial myocardial fibrosis compared to controls (Khan and Sheppard, Immunology, 118:10-24, 2006). As another example, the majority of cases of arrhythmogenic right ventricular cardiomyopathy (ARVC) in the United States have fat infiltration and scarring (fibrofatty ARVC) (Burke et al., Circulation, 97:1571-1580, 1998). In a study examining the histopathological characteristics of the ventricular myocardium in people with AKVC, it was found that extensive fibrosis was present in biopsy specimens from pediatric patients with AKVC (Nishikawa T. et al., Cardiovascular Pathology, vol. 8(4):185 -189, 1999).

Accordingly, in some embodiments, a method is provided for preventing, treating, reversing, suppressing, and/or reducing fibrosis and/or inflammation in a subject suffering from a cardiac or vascular disease or a disorder caused or complicated by fibrosis and/or inflammation, comprising administering to a subject in need thereof an inhibitory MASP-2

- 24 043102 agent, such as an inhibitory MASP-2 antibody. The method comprises administering a composition containing an amount of a MASP-2 inhibitor effective to suppress cardiac and/or vascular fibrosis and/or improve cardiac and/or vascular function.

In some embodiments, the present invention relates to a method of treating, suppressing, preventing, or attenuating fibrosis in a subject suffering from valvular fibrosis, comprising administering to the subject in need thereof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying cardiac or vascular disease or condition.

In some embodiments, the cardiac or vascular disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of cardiac fibrosis, myocardial infarction, atrial fibrosis, endomyocardial fibrosis, arrhythmogenic right ventricular cardiomyopathy (ARVC), vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis, and abdominal aortic aneurysm.

Chronic infectious diseases.

Chronic infectious diseases such as hepatitis C and hepatitis B cause tissue inflammation and fibrosis, and high activity of the lectin pathway can be harmful. In such diseases, MASP-2 inhibitors may be useful. For example, MBL and MASP-1 levels have been found to be important predictors of liver fibrosis severity in hepatitis C virus 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 potent activator of MASP-2 and the lectin pathway (Megyeri et al., J. Biol. Chem., 29:288(13):8922-34, 2013). Alphaviruses such as chikungunya virus and Ross River virus cause a strong inflammatory response in the host leading to arthritis and myositis, and this pathology is mediated by MBL and lectin pathway activation (Gunn et al., PLoS Pathog., 8(3):e1002586 , 2012).

Accordingly, in some embodiments, the disclosure provides a method for preventing, treating, reversing, suppressing, and/or reducing fibrosis and/or inflammation in a subject suffering from or having previously experienced a chronic infectious disease that causes inflammation and/or fibrosis, comprising administering to a subject in need thereof an inhibitory MASP-2 agent, such as a MASP-2 inhibitory antibody.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying chronic infectious disease.

In some embodiments, the chronic infectious disease that causes inflammation and/or fibrosis is selected from the group consisting of alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza.

Autoimmune diseases.

Scleroderma is a chronic autoimmune disease characterized by fibrosis, vascular changes, and autoantibodies. There are two main forms: limited systemic scleroderma and diffuse systemic scleroderma. Skin symptoms of localized systemic scleroderma affect the arms, shoulders, and face. Patients with this form of scleroderma often have one or more of the following complications: calcification, Raynaud's phenomenon, esophageal dysfunction, sclerodactyl, and telangiectasias. Diffuse systemic scleroderma is rapidly progressive and involves a large area of skin and one or more internal organs, often the kidneys, esophagus, heart, and/or lungs.

Scleroderma affects small blood vessels, known as arterioles, in all organs. First, arteriole endothelial cells die as a result of apoptosis along with smooth muscle cells. These cells are replaced by collagen and other fibrous material. Inflammatory cells, in particular CD4+ helper T cells, invade the arteriole and cause further damage.

Skin manifestations of scleroderma can be painful, lead to avoidance of use of the affected area (eg, hands, fingers, toes, toes, etc.) and can lead to disfigurement of such areas. The skin may develop ulcers, and such ulcers may be susceptible to infection or even gangrene. Ulcerated skin may be difficult to heal or heal slowly. Difficulty in healing skin ulcers can be particularly exacerbated in patients with circulatory problems such as Raynaud's phenomenon. In some embodiments, the methods of the present invention are used to treat scleroderma, such as the skin symptoms of scleroderma. In some embodiments, treatment of scleroderma includes treatment of skin ulcers, such as digital ulcers. Administration of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, can be used to reduce the fibrotic and/or inflammatory symptoms of scleroderma in affected tissues and/or organs.

In addition to skin symptoms/manifestations, scleroderma can also affect the heart, kidneys, lungs, joints, and digestive tract. In some embodiments, the implementation of the treatment of scleroderma includes the treatment of symptoms of the disease in any one or more of these tissues, for example, by reducing fibrotic and/or inflammatory symptoms. Lung-related problems are among the most serious complications of scleroderma and are responsible for most of the disease-related deaths. The two predominant lung conditions associated with scleroderma are pulmonary fibrosis and pulmonary hypertension. A patient with affected lungs may have either one or both of these conditions. Scleroderma-associated pulmonary fibrosis is one example of pulmonary fibrosis that can be treated with MASP-2 inhibitors. Scleroderma that affects the lung causes scarring (pulmonary fibrosis). This pulmonary fibrosis occurs in approximately 70% of patients with scleroderma, although its progression is usually slow and symptoms vary widely in severity. In patients who have symptoms associated with pulmonary fibrosis, such symptoms include dry cough, shortness of breath, and reduced exercise capacity. Approximately 16% of patients with some level of pulmonary fibrosis develop severe pulmonary fibrosis. In patients with severe pulmonary fibrosis, there is a significant decrease in lung function and alveolitis.

In some embodiments, the methods of the present invention are used to treat scleroderma, such as scleroderma-associated pulmonary fibrosis. The administration of MASP-2 inhibitory agents, such as MASP-2 inhibitory antibodies, can be used to reduce the fibrotic symptoms of scleroderma in the lungs. For example, these methods can be used to improve lung function and/or reduce the risk of death due to scleroderma.

In patients with scleroderma, the kidneys can also often be affected. Scleroderma-associated renal fibrosis is an example of renal fibrosis that can be treated by administering MASP-2 inhibitory agents such as anti-MASP-2 antibodies. In some embodiments, the methods of the present invention are used to treat scleroderma, such as scleroderma-associated renal fibrosis. In one embodiment, administration of MASP-2 inhibitory antibodies can be used to reduce the fibrotic symptoms of scleroderma in the kidney. For example, these methods can be used to improve kidney function, reduce protein in the urine, reduce the severity of hypertension, and/or reduce the risk of developing a kidney crisis, which can lead to fatal kidney failure.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder characterized by spontaneous B and T cell autoreactivity and multi-organ immune damage and can affect the skin, joints, kidneys, and other organs. Almost all people with SLE have joint pain, and most develop arthritis. Commonly affected joints are those of the fingers, hands, wrists, and knees. Common symptoms of SLE include arthritis; fatigue; general discomfort, restlessness, or a bad feeling (malaise); joint pain and swelling; muscle pain; nausea and vomiting; and skin rash. In addition, symptoms may also include abdominal pain; blood in the urine; fingers that change color when squeezed or in the cold; numbness and tingling; and red spots on the skin. Some patients with SLE have lung or kidney involvement. Without being limited by theory, inflammation and/or fibrosis in the lungs and kidneys leads to damage to these organs and the development of symptoms associated with damage to the lungs and/or kidneys. In some cases, patients with SLE develop a specific kidney condition called lupus nephritis. In some embodiments, the present invention relates to methods for treating SLE, comprising administering an effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody. Administration of MASP-2 inhibitory antibodies can be used to reduce one or more of the symptoms of SLE. In some embodiments, administration of anti-MASP-2 antibodies is used to treat SLE in a patient with lupus nephritis. In such cases, the treatment of SLE includes the treatment of lupus nephritis, for example, by reducing the symptoms of lupus nephritis. In some embodiments, the treatment includes treating the skin symptoms of SLE. In some embodiments, the implementation of the treatment includes reducing one or more symptoms of lupus nephritis. In some embodiments, the implementation of the treatment includes reducing, slowing down or eliminating the need for dialysis. In some embodiments, the implementation of the treatment includes reducing, slowing down or eliminating the need for kidney transplantation. In some embodiments, the treatment includes delaying or preventing the progression of lupus nephritis to renal failure or end-stage renal disease.

Lupus nephritis is an inflammation of the kidneys and a serious complication of systemic lupus erythematosus (SLE). As for the kidneys, lupus nephritis can lead to a disabling loss of kidney function. Patients with lupus nephritis may eventually develop kidney failure and may need dialysis or a kidney transplant. Associated complications that can also be treated with the methods of the invention include interstitial nephritis and nephrotic syndrome. Symptoms of lupus nephritis include blood in the urine, frothy urine, high blood pressure, protein in the urine, fluid retention, and swelling. Other symptoms include signs and symptoms of kidney fibrosis and/or kidney failure. If the disease is not treated, lupus nephritis can lead to kidney failure and even end stage kidney disease.

Accordingly, in some embodiments, a method is provided for preventing, treating, reversing, suppressing, and/or reducing fibrosis and/or inflammation in a subject suffering from an autoimmune disease that causes or is complicated by fibrosis and/or inflammation, comprising administering to a subject in need thereof an inhibitory MASP-2 an agent such as a MASP-2 inhibitory antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments suitable for the underlying autoimmune disease.

In some embodiments, the autoimmune disease that causes or is complicated by fibrosis and/or inflammation is selected from the group consisting of scleroderma and systemic lupus erythematosus (SLE).

Diseases and conditions of the central nervous system.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a disease or disorder of the central nervous system caused by or complicated by fibrosis and/or inflammation, comprising administering to the subject in need thereof an inhibitory MASP -2 agent, such as an anti-MASP-2 antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection either directly into the area of fibrosis or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or other parenteral route, or, in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying disease or disorder of the central nervous system.

In some embodiments, the central nervous system disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of stroke, traumatic brain injury, and spinal cord injury.

Skin diseases or conditions.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a skin disease or disorder caused by or complicated by fibrosis and/or inflammation, comprising administering to a subject in need thereof an inhibitory MASP-2 an agent such as a MASP-2 inhibitory antibody. This method comprises administering a composition containing an amount of a MASP-2 inhibitor effective

- 27 043102 effective for the suppression of fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition to the skin or topical application during surgery or local injection, either directly or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying skin disease or disorder.

In some embodiments, the skin disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of skin fibrosis, wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarring, and hypertrophic scars.

Diseases and conditions of bones and soft tissues of the musculoskeletal system.

In some embodiments, a method is provided for the prevention, treatment, management, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a bone or soft tissue disease or disorder caused or complicated by fibrosis and/or inflammation, comprising administering to the subject in need thereof an inhibitory MASP-2 agent, such as a MASP-2 inhibitory antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition to a bone or soft tissue structure, or locally during surgery or local injection, for example, directly or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments suitable for the underlying bone or soft tissue disease or disorder.

In some embodiments, the bone or soft tissue disease or disorder caused by or complicated by fibrosis and/or inflammation is selected from the group consisting of osteoporosis and/or osteopenia associated with e.g. cystic fibrosis, myelodysplastic conditions with increased bone fibrosis, adhesive capsulitis , Dupuytren's contracture and myelofibrosis.

Joint diseases or conditions.

In some embodiments, a method is provided for preventing, treating, reversing, suppressing, and/or reducing fibrosis and/or inflammation in a subject suffering from an articular disease or disorder caused or complicated by fibrosis and/or inflammation, comprising administering to a subject in need thereof an inhibitory MASP-2 an agent such as a MASP-2 inhibitory MASP-2 antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition to the joint, or local application during surgery or local injection, directly or remotely, for example, using a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying articular disease or disorder.

In some embodiments, the joint disease or disorder caused or complicated by fibrosis and/or inflammation is arthrofibrosis.

Diseases or conditions of the digestive system.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a disease or disorder of the digestive system caused or complicated by fibrosis and/or inflammation, comprising administering to the subject in need thereof an inhibitory MASP- 2 agents, such

- 28 043102 as MASP-2 inhibitory antibody MASP-2. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection, either directly or remotely, for example, via a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, anti-MASP-2 antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying disease or disorder of the digestive system.

In some embodiments, the disease or disorder of the digestive system caused by or complicated by fibrosis and/or inflammation is selected from the group consisting of Crohn's disease, ulcerative colitis, and pancreatic fibrosis.

Eye diseases or conditions.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from an eye disease or disorder caused or complicated by fibrosis and/or inflammation, comprising administering to a subject in need thereof an inhibitory MASP-2 an agent such as a MASP-2 inhibitory MASP-2 antibody. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection, either directly or remotely, for example, via a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application to the eye (for example, eye drops), or in the case of non-peptidergic agents, perhaps oral introduction. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying ocular disease or disorder.

In some embodiments, the ocular disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of anterior subcapsular cataract, posterior capsular opacification, macular degeneration, and retinal and vireal retinopathy.

Diseases or conditions of the reproductive organs.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a disease or disorder of the reproductive organs caused or complicated by fibrosis and/or inflammation, comprising administering to the subject in need thereof an inhibitory MASP- 2 agent such as MASP-2 inhibitory antibody MASP-2. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection, either directly or remotely, for example, via a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying reproductive disease or disorder.

In some embodiments, the disease or disorder of the reproductive organs caused or complicated by fibrosis and/or inflammation is selected from the group consisting of endometriosis and Peyronie's disease.

Scarring associated with trauma.

In some embodiments, a method is provided for preventing, treating, reversing, suppressing and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or condition resulting from trauma-related scarring, comprising administering to a subject in need thereof a MASP-2 inhibitory agent, such as the MASP-2 inhibitory antibody MASP-2. This method includes administering a composition containing an amount of an inhibitor

MASP-2 effective in suppressing fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection, either directly or remotely, for example, via a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or in the case of non-peptidergic agents, oral administration is possible. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying disease or disorder.

In some embodiments, trauma-related scarring is selected from the group consisting of surgical complications (e.g., surgical adhesions in which scar tissue can form between internal organs, causing contracture, pain, and can lead to infertility), chemotherapy drug-induced fibrosis, radiation-induced fibrosis and scarring associated with burns.

Additional diseases and disorders caused or complicated by fibrosis and/or inflammation.

In some embodiments, a method is provided for the prevention, treatment, relief, suppression and/or reduction of fibrosis and/or inflammation in a subject suffering from a disease or disorder caused and/or complicated by fibrosis and/or inflammation selected from the group consisting of organ transplantation, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, and pleural fibrosis, comprising administering to a subject in need thereof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody MASP-2. This method includes administering a composition containing an amount of a MASP-2 inhibitor effective to suppress fibrosis and/or inflammation.

The MASP-2 inhibitory composition can be administered locally to the area of fibrosis, for example, by topical application of the composition during surgery, or by local injection, either directly or remotely, for example, via a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application to the eye (for example, eye drops), or in the case of non-peptidergic agents, perhaps oral introduction. Administration may be repeated as determined by the physician until the condition is relieved or until control is achieved.

In some embodiments, MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying disease or disorder.

In some embodiments of any of the various methods and pharmaceutical compositions described herein, the MASP-2 inhibitory antibody selectively inhibits the lectin pathway without affecting the classical pathway.

In various aspects, the present invention relates to methods of suppressing the adverse effects of fibrosis and/or inflammation, including the introduction of a MASP-2 inhibitory agent in need of this subject. The MASP-2 inhibitory agents are 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, exemplary MASP-2 inhibitory agents include molecules that inhibit the biological activity of MASP-2 (such as small molecule inhibitors, anti-MASP-2 antibodies (for example, MASP-2 inhibitory antibodies), or blocking peptides that interact with MASP -2 or disrupt protein-protein interaction), and molecules that reduce MASP-2 expression (such as MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), thereby preventing MASP-2 2-activation of the lectin pathway of complement. MASP-2 inhibitory agents can be used alone as a primary therapy or in combination with other therapeutic agents as an adjuvant therapy to enhance the therapeutic effect of other medications.

Suppression of MASP-2 dependent complement activation is characterized by at least one of the following changes in a component of the complement system that result from administration of an inhibitory MASP-2 agent in accordance with the methods of the invention: suppression of the generation or production of MASP-2 dependent complement activation products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2), reduction in C4 degradation and C4b deposition (measured, for example, as described in Example 2), or reduction in C3 degradation and deposits of C3b (measured, for example, as described in example 2).

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In accordance with the present invention, MASP-2 inhibitory agents are used that are effective in suppressing fibrosis and/or inflammation and exhibit detectable anti-fibrotic activity and/or induce a reduction in fibrosis. In the context of the invention, the antifibrotic activity may include at least one or more of the following:

(1) reduction of inflammation, for example, as measured by the activation and recruitment of macrophages and endothelial cells; recruitment and activation of lymphocytes and/or eosinophils by secretion of a number of cytokines/chemokines; release of cytotoxic mediators and fibrogenic cytokines;

(2) reduced cell proliferation, ECM synthesis or angiogenesis; and/or (3) a decrease in the level of collagen deposition compared to fibrotic activity in the absence of an inhibitory MASP-2 agent.

Evaluation of an anti-fibrotic agent, such as a MASP-2 inhibitory agent, can be performed by detection by any method known to one of skill in the art. For example, the evaluation of an anti-fibrotic agent can be performed using the UUO model (as described in the present application in examples 12 and 14). If the anti-fibrotic activity and/or reduction or reduction of fibrosis being assessed is assessed with a MASP-2 inhibitory agent, the MASP-2 inhibitory agent is considered to be used as a drug for preventing, treating, reversing and/or suppressing fibrosis.

Fibrosis assessment can be performed periodically, for example, every week or every month. Thus, the increase/decrease in fibrosis and/or the presence of antifibrotic activity can be assessed periodically, for example, every week or month. This assessment is preferably performed at multiple time points for a given subject, or at one or more time points for a given subject and a healthy control. The evaluation may be carried out at regular intervals, such as every week or every month. Therefore, the evaluation can be carried out regularly, for example, every week or every month. If a decrease in fibrosis or the presence of antifibrotic activity is detected as a result of the evaluation, the MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, is considered to exhibit detectable antifibrotic activity and/or induce a decrease or decrease in fibrosis.

MASP-2 inhibitory agents in the practice of this aspect of the invention include, for example, MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, soluble MASP-2 receptors, and expression inhibitors. MASP-2 inhibitory agents can suppress the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, an inhibitory agent can effectively block the interaction of a MASP-2 protein with a protein, interfere with MASP-2 dimerization or assembly, block Ca ²⁺ binding, interfere with the active site of a MASP-2 serine protease, or decrease the level of expression of a MASP-2 protein.

In some embodiments, MASP-2 inhibitory agents selectively suppress MASP-2-dependent complement activation without affecting the functional activity of the C1q-dependent complement activation system.

In one embodiment, the MASP-2 inhibitory agent used in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO: 6 with a binding affinity of at least 10 times binding affinity for other antigens in the complement system. In another embodiment, the MASP-2 inhibitory agent specifically binds to a polypeptide comprising SEQ ID NO: 6 with a binding affinity of at least 100 times that of other antigens in the complement system. In one embodiment, the MASP-2 inhibitory agent specifically binds to at least one of (i) the CCP1-CCP2 domain (aa 300-431 in SEQ ID NO: 6); or (ii) the MASP-2 serine protease domain (aa 445-682 in SEQ ID NO: 6), and inhibits the activation of MASP-2 dependent complement.

In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to MASP-2. The binding affinity of the MASP-2 inhibitory agent can be determined using an appropriate binding assay.

The MASP-2 polypeptide has a molecular structure similar to that of 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, forming the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIG. 2, the human MASP-2 gene contains twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. Alternative splicing produces a 20 kDa protein called MBL-associated protein 19 (MAp19, also called sMAP ) (SEQ ID NO: 2) encoded by (SEQ ID NO: 1) derived from exons B, C, D, and E as shown in FIG. 2. The cDNA molecule shown in SEQ ID NO: 50 encodes mouse MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO: 51) and provides a mouse MASP-2 polypeptide with a leader sequence that is cleaved after secretion to form mature form of mouse 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 a rat MASP-2 polypeptide with a leader sequence that is cleaved to form the mature form after secretion. rat MASP-2 (SEQ ID NO: 55).

Those skilled in the art will appreciate that the sequences disclosed in SEQ ID NO: 4, SEQ ID NO: 50, and SEQ ID NO: 53 represent single alleles of human, mouse, and rat MASP-2, respectively, and that allelic variants and alternative splicing. The allelic variants of the nucleotide sequences set forth in SEQ ID NO: 4, SEQ ID NO: 50, and SEQ ID NO: 53, including those containing silence mutations and those in which mutations result in amino acid sequence changes, are within the scope of this invention. Allelic variants of the MASP-2 sequence can 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 FIG. 1 and 2A and include the sea urchin N-terminal C1r/C1s/VEGF/bone morphogenetic protein (CUBI) domains (aa 1-121 in SEQ ID NO: 6), epidermal growth factor like domain (aa 122-166), the second CUBI domain (aa 167-293), as well as a tandem of complement regulatory protein domains. Alternative splicing of the MASP-2 gene results in the formation of MAp19 shown in FIG. 1. MAp19 is a non-enzymatic protein containing the N-terminal CUBI-EGF region of MASP-2 with four additional residues (EQSL) derived from the E exon shown in FIG. 1.

Several proteins have been shown to be able to bind or interact with MASP-2 through protein-protein interactions. For example, MASP-2 is known to be able to bind and form Ca ²⁺ -dependent complexes with the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin protein complex has been shown to be able to activate complement through MASP-2-dependent cleavage of C4 and C2 proteins (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 domains of MASP-2 are important for the binding of MASP-2 to MBL (Thielens, NM et al., J. Immunol., 166:5068, 2001). The CUBIEGFCUBII domains have also been shown to mediate MASP-2 dimerization required for active MBL complex formation (Wallis, R. et al., J. Biol. Chem., 275:30962-30969, 2000). It follows that MASP-2 inhibitory agents are characterized by their ability to bind or interact with MASP-2 target sites that play an important role in the process of MASP-2-dependent complement activation.

Ahtu-MASP-2 antibodies.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that suppresses the MASP-2 dependent complement activation system. Ahtu-MASP-2 antibodies useful in this aspect of the present invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody-producing mammal, and these antibodies can be multispecific, chimeric, humanized, anti-idiotypic antibodies and antibody fragments. Antibody fragments include Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv fragments, scFv fragments, and single chain antibodies described below.

Ahtu-MASP-2 antibodies can be screened for suppression of the MASP-2-dependent complement activation system and for antifibrotic activity and/or suppression of proteinuria-associated renal injury or adriamycin-induced nephropathy using the assay described herein. Several anti-MASP-2 antibodies have been described in the scientific literature, several of which are listed in Table. 1. For example, as shown in the present application in examples 10 and 11, it was found that anti-MASP-2 antibodies Fab2 block MASP-2-dependent complement activation. As shown in Example 12 and described in WO2012/151481, incorporated herein by reference, fully human anti-MASP-2 scFv antibodies (eg, OMS646) were determined to block MASP-2 dependent complement activation. As described in Example 13, as well as in WO 024141442, incorporated herein by reference, anti-MASP-2 antibodies bearing the SGMI-2 peptide and fragments thereof exhibiting MASP-2 inhibitory activity were obtained by fusion with the amino acid the sequence of the SGMI-2 peptide (SEQ ID NO: 72, 73 or 74) at the amino or carboxyl terminus of the heavy and/or light chain of a human anti-MASP-2 antibody (eg, OMS646-SGMI-2).

Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises a human antibody such as, for example, OMS646. Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the compositions and methods of the claimed invention comprises a human antibody that binds a polypeptide consisting of human MASP-2 (SEQ ID NO: 6), wherein the antibody contains ( I) (a) a heavy chain variable region containing

i) a heavy chain CDR-H1 containing the amino acid sequence from aa 31-35 to

SEQ ID NO: 67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from aa 50-65 in SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from aa 95-107 in SEQ ID NO: 67; And

b) a light chain variable region containing

i) a light chain CDR-L1 containing the amino acid sequence from aa 24-34 in SEQ ID NO: 69; and ii) a light chain CDR-L2 comprising the amino acid sequence from aa 50-56 in SEQ ID NO: 69; and iii) a light chain CDR-L3 comprising the amino acid sequence from aa 89-97 in SEQ ID NO: 69; or (ii) a variant thereof comprising a heavy chain variable region with at least 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) with SEQ ID NO: 67; and a light chain variable region with at least 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) with SEQ ID NO: 69.

In some embodiments, the method comprises administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof 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 shown in SEQ ID NO: 69.

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

Table 1

Exemplary Masp-2 Specific Antibodies

Antigen Type of antibody Link Recombinant MASP-2 Rat polyclonal Peterson, S.V., et al., Mol. Immunol. 37:803811, 2000 Fragment of recombinant human CCP1/2-SP (MoAb 8B5) Rat IgGl) MoAb (subclass Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003 Recombinant human MAR19 (MoAb 6G12) (cross-reaction with MASP- 2) Rat IgGl) MoAb (subclass Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003

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hMASP-2 Mouse MoAb (S/P) Mouse MoAb (N-term) Peterson, S.V., et al., Mol. Immunol. 35:409 April 1998 hMASP-2 (domain CCP1-CCP2-SP) rat MoAb: NimoablOl produced by hybridoma cell line 03050904 (ECASS) WO2004/106384 hMASP-2 (full size, murine MoAbs: NimoAbl04, WO2004/106384 hi s-labeled) produced by hybridoma cells line M0545YM035 (DSMZ) NimoAblOS, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAbl09 produced by hybridoma cell line M0545YM046 (DSMZ) NimoAbllO produced by hybridoma cell line M0545YM048 (DSMZ) Rat MASP-2 (full size) fragments of anti-MARP-2 Eab2 antibodies Example 10 hMASP-2 (full size) Fully human scFv clones Example 12 and WO2012/151481 hMASP-2 (full size) Anti-MAP-2 antibodies, carrying the SGMI-2 peptide Example 13 and WO2014/144542

Ahtu-MASP-2 antibodies with reduced effector function.

In some embodiments of this aspect of the present invention, the anti-MASP-2 antibodies have reduced effector function to reduce inflammation that can occur when the classical complement pathway is activated. The ability of IgG molecules to trigger the classical complement activation pathway has been found to be associated with the Fc region of the molecule (Duncan, A.R. et al., Nature, 332:738-740, 1988). IgG molecules from which the Fc region has been removed by enzymatic cleavage lack this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be obtained by removing the Fc region of a molecule containing a genetically engineered Fc sequence that has minimal effector function, this molecule can be of both the human IgG2 isotype and the IgG4 isotype.

Antibodies with reduced effector function can be generated using standard molecular biological manipulations of the Fc region of IgG heavy chains as described herein and shown by Jolliffe et al., Int'l Rev. Immunol. 10:241-250, 11993; and Rodrigues et al., J. Immunol., 151:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that have a 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 complete human antibodies specific for human MASP-2, including IgG2 or IgG4 isotypes, can be generated by one of several methods known to one of skill in the art, as described by Vaughan, T.J. et al., Nature Biotechnical, 16:535-539, 1998.

Obtaining anti-MASP-2 antibodies.

Ahtu-MASP-2 antibodies can be generated using MASP-2 polypeptides (eg, full-length MASP-2) or antigenic peptides bearing a MASP-2 epitope (eg, a portion of a MASP-2 polypeptide). Immunogenic peptides can consist of as few as five amino acid residues. For example

- 34 043102, a MASP-2 polypeptide containing the complete amino acid sequence of SEQ ID NO: 6 can be used to induce anti-MA8P-2 antibodies that can be used in the method of the present invention. It is known that specific MASP-2 domains involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as the serine protease active site region, can be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides containing a region of at least 6 amino acids of a MASP-2 polypeptide (SEQ ID NO: 6) can also be used to induce MASP-2 antibodies. Additional examples of the obtained MASP-2 antigens with the ability to induce MASP-2 antibodies are shown below in table. 2. The MASP-2 peptides and polypeptides used to generate antibodies can be isolated as natural polypeptides or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides such as MASP-2A, as described hereinafter. In some embodiments of this aspect of the present invention, anti-MA8P-2 antibodies are obtained using a transgenic mouse line, as described below in this application.

Antigens capable of producing anti-MA8P-2 antibodies also include fusion polypeptides, for example, polypeptides resulting from the 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 a portion of the polypeptide is a hapten, that portion may advantageously be attached to or coupled to a macromolecular carrier (such as keyhole hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

table 2

MASP-2 antigens

SEQID NO: Amino acid sequence SEQ ID NO: 6 Human MASP-2 protein SEQ ID NO: 51 Mouse protein MASP-2 SEQ ID NO: 8 CUBI domain of human MASP-2 (aa 1-121 in SEQ ID NO:6) SEQ ID NO: 9 CUBIEGF domains of human MASP-2 (aa 1-166 in SEQ ID NO:6) SEQ ID NO: 10 Human CUBIEGFCUBII domains MASP-2 (aa 1-293 in SEQ ID NO:6) SEQ ID NO: 11 Human MASP-2 EGF domain (aa 122-166 in SEQ ID NO:6) SEQ ID NO: 12 Serine protease domain human MASP-2 (aa 429-671 in SEQ ID NO:6) SEQ ID NO: 13 GKDSCRGDAGGALVFL Mutant form with an inactivated serine protease domain

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(aa 610-625 in SEQ ID NO:6 with mutant Ser 618) SEQ ID NO: 14 TPLGPKWPEPVFGRL Human peptide CUBI SEQ ID NO: 15: TAPPGYRLRLYFTHFDLEL SHLCEYDFVKLSSGAKVLATLCGQ Human peptide CUBI SEQ ID NO: 16: TFRSDYSN MBL-binding site in human CUBI domain SEQ ID NO: 17: FYSLGSSLDITFRSDYSNEKPFTG F MBL-binding site in human CUBI domain SEQ ID NO: 18 IDECQVAPG EGF peptide SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGAL V Peptide from the active site of serine protease

polyclonal antibodies.

Polyclonal anti-MA8P-2 antibodies can be prepared by immunizing animals with the MASP-2 polypeptide or immunogenic portion thereof, by methods well known to one of skill in the art. See, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), p. 105. The immunogenicity of a MASP-2 polypeptide can be increased by the use of an adjuvant, including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysolecithin, high molecular weight alcohols of pluronics, polyanions, oil emulsions, fissurela hemocyanin and dinitrophenol. Animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep are commonly used to produce polyclonal antibodies. Alternatively, the anti-MA8P-2 antibody used in the present invention can also be obtained from a great ape. A general method for generating diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Application Publication WO 91/11465, and in Losman, M.J. et al., Int. J. Cancer, 46:310, 1990. Serum containing immunologically active antibodies is then obtained from the blood of such immunized animals using standard well known procedures.

monoclonal antibodies.

In some embodiments, the MASP-2 inhibitory agent is an anti-MA8P-2 monoclonal antibody. Monoclonal Ahth-MASP-2 antibodies are highly specific epitope directed against a single epitope of MASP-2. As used herein, the modifier monoclonal indicates the nature of the antibody derived from a substantially homogeneous population of antibodies, and does not imply the production of antibodies by any particular method. Monoclonal antibodies can be produced by any method that produces antibody molecules in culture of a stable cell line, such as, for example, the hybridoma method described in Kohler, G. et al., Nature, 256:495, 1975, or they can be obtained by methods recombinant DNA (see, for example, US patent 4816567, Cabilly). Monoclonal antibodies can also be isolated from an antibody phage library using the methods 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 belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (eg, BALB/c mice) with a composition containing a MASP-2 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are isolated from the mouse and resuspended in culture medium. The splenocytes are then fused with an immortalized cell line to form a hybridoma. The resulting hybridomas are grown in cell culture and screened for their ability to produce anti-MASP-2 monoclonal antibodies. Further in this description, examples of obtaining monoclonal anti-MA8P-2 antibodies are given (see also Current Protocols in Immunology, vol. 1, John Wiley & Sons, p. 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies can be obtained from transgenic mice that are genetically engineered to produce human antibodies in response to an antigenic stimulus. In this method, human immunoglobulin heavy and light chain locus elements are introduced into lines

-36043102 mice derived from cell lines of embryonic stem cells that contain 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 antigens described in the present invention, and these mice can be used to generate anti-human MASP-2 antibody-secreting hybridomas by fusing B cells derived from such animals, with suitable myeloma cell lines by the usual method of Koehler-Milstein, as described below in this application. Transgenic mice with the human immunoglobulin genome are commercially available (eg, from Abgenix, Inc., Fremont, C.A.; Medarex, Inc., Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described in the scientific literature, for example 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 culture by various well known methods. Such isolation methods include protein A sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan, p. 2.7.1-2.7.12, p. 2.9.1-2.9.3; Baines et al., Purification of Immunoglobulin G (IgG), Methods in Molecular Biology, The Humana Press, Inc., vol.10, p.79-104, 1992).

Upon receipt, polyclonal, monoclonal, or phage-derived antibodies are first tested for specific binding to MASP-2. To determine antibodies that specifically bind to MASP-2, you can use many different methods known to the person skilled in the art. Illustrative examples of assays performed by standard methods include Western blot or immunoprecipitation assay (e.g., as described by Ausubel et al.), immunoelectrophoresis, enzyme immunoassay (ELISA), dot blot, suppression assay, or competition assay (as described by Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies capable of specifically binding to MASP-2 have been identified, anti-MASP-2 antibodies are tested for their ability to function as an inhibitory agent of MASP-2 by one of several assessment methods, such as, for example, the C4 lectin-specific cleavage assay (described in Example 2), C3b deposition analysis (described in Example 2) or C4b deposition analysis (described in Example 2).

The binding affinity of anti-MASP-2 monoclonal antibodies can be readily determined by those skilled in the art (see, for example, Scatchard, A., NY Acad. Sci., 51:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies used in the methods of the present invention bind to MASP-2 with a binding affinity of <100 nmol, preferably <10 nmol, and more preferably <2 nmol.

Chimeric/humanized antibodies.

Monoclonal antibodies used in the method of the present invention include chimeric antibodies in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder chain(s) is identical or homologous to the corresponding sequences of antibodies isolated from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies (US patent 4816567, Cabilly; Morrison, S.L. et al., Proc. Nat'l Acad. Sci., USA, 81:6851-6855, 1984).

One form of the chimeric antibody used in the present invention is a humanized anti-MASP-2 monoclonal antibody. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring non-human (eg, murine) complementarity-determining regions (CDRs) from mouse immunoglobulin heavy and light chain variable regions into the human variable domain. Typically, human antibody residues are then replaced in the framework regions of the non-human prototypes. Moreover, humanized antibodies may contain residues that are not found in recipient antibodies or donor antibodies. These modifications are made to further improve antibody performance. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains wherein 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 from human immunoglobulin sequences. The humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. A detailed description can be found in Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; 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 site of CDRH3. In addition, the Fc regions can be substituted such that IgA or IgM as well as human IgG antibodies are produced. Thus, humanized antibodies will find particular clinical utility, as they will specifically recognize human MASP-2 without eliciting an immune response in humans against the antibody itself. As a consequence, they are better suited for in vivo administration to humans, especially when repeated or prolonged administration is required.

An example of making a humanized anti-MASP-2 antibody from a mouse monoclonal anti-MASP-2 antibody is given in Example 6. Methods for making humanized monoclonal antibodies are also described in, for example, 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 (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., p. 399-434, 1996; and US Pat. No. 5,693,762 to Queen, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific antibody regions, such as Protein Design Labs (Mountain View, CA).

recombinant antibodies.

Ahtu-MASP-2 antibodies can also be obtained using recombinant methods. For example, human antibodies can be generated using a human immunoglobulin expression library (available, for example, from Stratagene, Corp., La Jolla, CA) to generate human antibody fragments (VH, VL, Fv, Fd, Fab or F(ab') ₂ ). These fragments are then used to construct complete human antibodies using methods similar to those used to generate chimeric antibodies.

Anti-idiotypic antibodies.

Once anti-MASP-2 antibodies with the desired inhibitory activity have been identified, these antibodies can be used to generate anti-idiotypic antibodies that are similar to the MASP-2 portion by methods well known in the art. See, for example, Greenspan, M.S. et al., FASEB J., 7:437, 1993. For example, antibodies that bind to MASP-2 and competitively repress MASP-2 protein interactions required for complement activation can be used to generate anti-idiotypic antibodies that have similarities to the MBL-binding site of the MASP-2 protein and therefore bind to and neutralize the binding of MASP-2 ligands such as, for example, MBL.

Fragments of immunoglobulin.

The MASP-2 inhibitory agents used in the method of the invention encompass not only intact immunoglobulin molecules, but well known fragments including Fab, Fab', F(ab) ₂ , F(ab') ₂ and Fv fragments, scFv- fragments, dimers, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

It is well known that only a small portion of an antibody molecule, the paratope, is involved in the binding of an antibody to its epitope (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of the antibody are effectors of the classical complement pathway but are not involved in antigen binding. An antibody from which a pFc' region has been enzymatically cleaved or which has been produced without a pFc' region is referred to as the F(ab') ₂ fragment and contains both antigen-binding regions of the intact antibody. The isolated fragment F(ab') ₂ refers to a bivalent monoclonal fragment due to the presence of two antigen-binding sites. Similarly, an antibody from which an Fc region has been enzymatically cleaved, or which has been produced without an Fc region, is designated a Fab fragment and contains one of the antigen-binding regions of the intact antibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis, such as pepsi or papain hydrolysis of whole antibodies, by standard methods. For example, antibody fragments can be prepared by enzymatic digestion of antibodies with pepsin to produce a 5S fragment, designated F(ab')2. This fragment can be further cleaved with a thiol reducing agent to give 3,5S Fab' monovalent fragments. Optionally, the cleavage reaction can be carried out using a blocking group for sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic digestion with pepsin yields two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in US patent 4331647, 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 in Coligan, p. 2.8.1-2.8.10, 2.10-2.10.4.

In some embodiments, the use of antibody fragments lacking an Fc region is preferred because it avoids activation of the classical complement pathway that is initiated by Fc binding to the Fcy receptor. There are several ways to produce MoAb that do not affect Fcy receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed by partial cleavage involving proteolytic enzymes (eg, ficin cleavage) to produce, for example, antigen-binding antibody fragments such as Fab or F(ab) ₂ fragments (Mariani, M. et al., Mol. Im-38 043102 munol. 28:69-71, 1991). Alternatively, the γ4 human IgG isotype that does not bind to Fcy receptors can be used to generate a humanized antibody as described herein. Antibodies, single-chain antibodies, and antigen-binding domains that lack

The Fc domain can also be created by the recombinant methods described in this application.

Fragments of single-chain antibodies.

Alternatively, one can create MASP-2-specific molecules that bind a single polypeptide chain, in which the Fv regions of the heavy and light chains are linked. Fv fragments can be linked via a peptide linker to form a single chain antigen binding protein (scFv). Such single-stranded antigen-binding proteins are produced by generating a structural gene containing DNA sequences encoding the VH and VL domains associated with the oligonucleotide. The structural gene is inserted into an expression vector which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a peptide linker linking two V domains. Methods for preparing scFv are described, for example, in Whitlow et al., Methods: A Companion to Methods in Enzymology, 2:97, 1991; Bird et al., Science, 242:423, 1988; U.S. Pat. No. 4,946,778 Ladner; Pack, P. et al., Bio/Technology, 11:1271, 1993.

As an illustrative example, a MASP-2-specific scFv can be generated by exposing lymphocytes to a MASP-2 polypeptide in vitro and selecting antibody libraries by phage display in phages or similar vectors (e.g., by using immobilized or labeled MASP-2 proteins or peptides). ). Genes encoding polypeptides containing potential MASP-2 polypeptide binding domains can be obtained by screening any peptide libraries displayed on phage or bacteria, such as E. coli. These random peptide phage libraries can be used to screen for peptides that interact with MASP-2. Methods for generating and screening such random peptide display libraries are well known (see US Pat. No. 5,223,409 to Lardner; US Pat. No. 4,946,778 to Ladner; US Pat. No. 5,403,484 to Lardner; US Pat. Academic Press, Inc., 1996), and random peptide display libraries and screening kits for such libraries are commercially available from, for example, 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 used in this aspect of the present invention is a peptide encoding a single complementarity determining region (CDR) that binds to an epitope on the MASP-2 antigen and suppresses MASP-2 dependent complement activation. CDR peptides (minimum recognition units) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are obtained, for example, by polymerase chain reaction to synthesize a variable region from the RNA of an antibody-producing cell (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106, 1991; Courtenay-Luck, Genetic Manipulation of Monoclonal Antibodies, Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al (eds.), p.166, Cambridge University Press, 1995 and Ward et al., Genetic Manipulation and Expression of Antibodies, Monoclonal Antibodies : Principles and Applications, Birch et al (eds.), p.137, Wiley-Liss, Inc., 1995).

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

peptide inhibitors.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises isolated peptide inhibitors of MASP-2, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2 dependent complement activation system. As used herein, the term isolated MASP-2 peptide inhibitors refers to peptides that inhibit MASP-2-dependent complement activation by binding to MASP-2, competing with MASP-2 for binding to another recognition molecule (e.g., MBL, H -ficolin, M-ficolin or L-ficolin) by activating the lectin pathway and/or by interacting directly with MASP-2, thus suppressing MASP-2 dependent complement activation, while these inhibitors are essentially pure and contain virtually no other substances, with which they can be found under natural conditions, to the extent that they are suitable for their intended use.

Peptide inhibitors have been successfully used in vivo to suppress protein-protein interactions and the catalytic site. For example, peptide adhesion molecule inhibitors structurally related to LFA-1 have recently been approved for clinical use in coagulopathy (Ohman, E.M. et al., European Heart J., 16:50-55, 1995). Short linear peptides (<30 amino acids) have been shown to be able to prevent or interfere with integrin-dependent adhesion (Murayama, O. et al., J. Biochem., 120:445-51, 1996). Longer peptides of 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 lower dissociation rates than shorter peptides and therefore may be more effective inhibitors. Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrins in vivo in the treatment of human inflammatory diseases (Jackson, D.Y. et al., J. Med. Chem., 40:3359-68, 1997). One way to make cyclic peptides involves synthesis of peptides in which the terminal amino acids of the peptide are cysteines, which allows the peptide to exist in a cyclic form through the formation of disulfide bonds between the terminal amino acids, which has been shown to improve binding affinity and in vivo half-life during treatment. hematopoietic neoplasia (for example, US patent 6649592, Larson).

Synthetic peptide inhibitors of MASP-2.

The MASP-2 inhibitory peptides used in the methods of this aspect of the present invention are amino acid sequences that mimic target sites important for MASP-2 function. The sizes of inhibitory peptides used in practice in the methods of the present invention range from about 5 amino acids to about 300 amino acids. In table. 3 is a list of exemplary inhibitory peptides that may be used in the practice of this aspect of the present invention. A candidate MASP-2 inhibitory peptide can be tested for its ability to function as a MASP-2 inhibitory agent in one or more assays, including, for example, the C4 lectin-specific cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2). 2).

In some embodiments, MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full-length mature MASP-2 protein (SEQ ID NO: 6) or from a specific domain of the MASP-2 protein, such as, for example, the CUBI domain (SEQ ID NO: 8), a CuBIEGF domain (sEq ID NO: 9), an EGF domain (SEQ ID NO: 11), and a serine protease domain (SEQ ID NO: 12). As previously described, CUBEGFCUBII regions have been shown to be required for dimerization and binding to MBL (Thielens et al., supra). In particular, in a study to identify a human carrying a homozygous Asp105 to Gly105 substitution resulting in the loss of MASP-2 from the MBL complex, it was shown that the peptide sequence TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 is involved in binding with MBL (Stengaard-Pedersen, K. et al., New England J. Med., 349:554-560, 2003).

In some embodiments, MASP-2 inhibitory peptides are derived from lectin peptides that bind to MASP-2 and are involved in activation of the lectin complement pathway. Several different lectins have been identified that are involved in this pathway, including mannan-binding lectin ((MBL), L-ficolin, M-ficolin, and H-ficolin (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 or homotrimeric subunits, each having N-terminal collagen-like fibers, with carbohydrate recognition domains These different lectins have been shown to bind to MASP-2 and the lectin/MASP-2 complex activates complement via cleavage C4 and C2 proteins.H-ficolin has a 24 amino acid terminal region, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a 12-amino acid neck domain, and a 207-amino acid fibrinogen-like domain (Matsushita, M. et al., J Immunol., 168:3502-3506, 2002. H-ficolin binds to GlcNAc and agglutinates LPS-derived human erythrocytes derived from S. typhimurium, S. minnesota and E. coli. H-ficolin has been shown to bind to MASP-2 and MAp 19 and activate the lectin complement pathway. Id. L-ficolin/P35 also binds to GlcNAc and has been shown to bind to MASP-2 and MAp 19 in human serum, and this complex activates the complement lectin pathway (Matsushita, M. et al., J. Immunol., 164 :2281, 2000). Accordingly, the MASP-2 inhibitory peptides used in the present invention may comprise a region of at least 5 amino acids selected from MBL protein (SEQ ID NO: 21), H-ficolin protein (Genbank accession number NM_173452), M -ficolin (Genbank accession number 000602) and protein L-ficolin (Genbank accession number NM_015838).

More specifically, scientists have identified a MASP-2 binding site in MBL consisting of 12 Gly-X-Y GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POG NOG PSG SOG PKG QKG DOG KS triplets (SEQ ID NO: 26) that are located between the hinge and cervical regions at the C-terminus of the MBP collagen-like domain (Wallis, R et al., J. Biol. Chem., 279:14065, 2004). This MASP-2 binding site is also very conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in all three lectin proteins, containing the amino acid sequence OGK-X-GP (SEQ ID NO: 22), where the letter O means hydroxyproline and the letter X represents a hydrophobic residue (Wallis et al., 2004, supra). Accordingly, in some embodiments, the MASP-2 inhibitory peptides used in this aspect of the present invention are at least 6 amino acids in length and

- 40 043102 contain SEQ ID NO: 22. MBL-derived peptides that include the amino acid sequence GLR GLQ GPO GKL GPO G (SEQ ID NO: 24) have been shown to bind to MASP-2 in vitro (Wallis et al. , 2004, supra). To enhance binding to MASP-2, peptides flanked by two GPO triplets at each end (GPO GPO GLR GLQ GPO GKL GPO GGP OGP O, SEQ ID NO: 25) can be synthesized to increase the likelihood of triple helix formation found in native MBL protein ( as described in Wallis, R. et al., J. Biol. Chem., 279:14065, 2004).

MASP-2 inhibitory peptides can also be derived from human H-ficolin, 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 site in H-ficolin. Also included are peptides derived from human L-ficolin, which includes 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 site in L-ficolin.

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

Table 3

Exemplary MASP-2 inhibitory peptides

SEQID NO Source SEQ ID NO: 6 Human MASP-2 protein SEQ ID NO: 8 CUBI MASP-2 domain (aa 1-121 in SEQ ID NO: 6) SEQ ID NO: 9 CUBIEGF MASP-2 domain (aa 1-166 in SEQ ID NO: 6) SEQ ID NO: 10 CUBIEGFCUBII MASP-2 domains (aa 1-293 in SEQ ID NO:6) SEQ ID NO: 11 EGF MASP-2 domain (aa 122-166) SEQ ID NO: 12 MASP-2 serine protease domain (aa 42 9671) SEQ ID NO: 16 MBL-binding site in MASP-2 SEQ ID NO: 3 Human MApl9 SEQ ID NO: 21 Human protein MBL SEQ ID NO: 22 OGK-X-GP, Where "o" is hydroxyproline, and "X" is hydrophobic amino acid residue Synthetic peptide, consensus binding site from human MBL and human ficolins SEQ ID NO: 23 OGKLG The main part of the binding site human MBL SEQ ID NO: 24 GLR-GLQ-GPO-GKL-GPO-G Human ICBM triplets 6-10- demonstrating binding to MASP-2 SEQ ID NO: 25 GPOGPOGLRGLQGPOGKLGPOG GPOGPO Human MBR triplets with GPO added to enhance triple helix formation SEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGL QGPOGKLGPOGNOGPSGSOGPK GQKGDOGKS Human ICBM triplets 1-17 SEQ ID NO: 27 GAOGSOGEKGAOGPQGPOGPOG KMGPKGEOGDO Human H-ficolin (Hataka)

- 41 043102

SEQ ID NO: 28 GCOGLOGAOGDKGEAGTNGKRG ERGPOGPOGKAGPOGPNGAOGEO Human L-ficolin P35 SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI Human C4 cleavage site SEQ ID NO: 72 LEVTCEPGTTFKDKCNTCRCGSDG KSAVTKTKLWCNQ SGMI-2L (full size) SEQ ID NO: 73 TCEPGTTFKDKCNTCRCGSDGKSA VCTKLWCNQ SGMI-2M (short version, medium length) SEQ ID NO: 74 TCRCGSDGKSAVCTKLWCNQ SGMI-2S (short variant, short) Note: The letter "O" means hydroxyproline, the letter "X" means a hydrophobic residue.

Peptides derived from the C4 cleavage site, as well as other MASP-2 serine protease site inhibitory peptides, can be chemically modified to become irreversibly potent protease inhibitors. For example, suitable modifications may include, without limitation, halomethyl ketones (Br, Cl, I, F) at the C-terminal end, Asp, or Glu, or may be attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxide or imine containing groups on amino or carboxyl terminal ends or functional side chains; or esters, imidates at amino or carboxyl terminals or functional side chains. Such modifications may provide an advantage in irreversible inhibition of the enzyme by covalent attachment of the peptide. This may lead to a reduction in the effective dose and/or a decrease in the frequency of administration of the peptide inhibitor.

In addition to the inhibitory peptides described above, the MASP-2 inhibitory peptides used in the method of the present invention include peptides containing the MASP-2 CDR3 binding site in the anti-MASP-2 MoAb produced by the method described herein. The sequence of CDR regions for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is a peptide that is typically between 100 and 150 amino acids in length. The light chain variable region is a peptide that is typically between 80 and 130 amino acids in length. CDR sequences within the heavy and light chain variable regions include sequences of only about 3-25 amino acids, which can be readily sequenced by one of skill in the art.

Those skilled in the art will recognize that substantially homologous variants of the MASP-2 inhibitory peptides described above will also exhibit MASP-2 inhibitory activity. Illustrative options include, without limitation, peptides containing insertions, deletions, substitutions, and/or additional amino acids at the carboxy-terminal or amino-terminal portions of the claimed peptides, and mixtures thereof. Accordingly, such homologous peptides having MASP2 inhibitory activity are also contemplated for use in the methods of the present invention. The described peptides may also include repetitive motifs and other modifications with conservative substitutions. Conservative variants are described herein and include substitution of one amino acid for another amino acid with a similar charge, similar size, similar hydrophobicity, etc.

MASP-2 inhibitory peptides can be modified to increase solubility and/or maximize positive or negative charge to enhance segment similarity in intact protein. The derivative may or may not have the exact primary amino acid structure of the peptide disclosed in this application, provided that the derivative remains functionally active against MASP-2 inhibition. Modifications may include replacing an amino acid with one of the well-known twenty amino acids, or any other amino acid, derivatized or substituted with additional desired characteristics, such as resistance to enzyme degradation, or a D-amino acid, or substitution with another molecule or compound, such as a carbohydrate, that mimics natural the conformation and function of the amino acid, amino acids, or peptide; amino acid deletion; insertion of one of the twenty well-known amino acids or any other amino acid derivatized or substituted

- 42 043102 amino acids with additional desired characteristics such as resistance to enzymatic degradation, or D-amino acids, or replacement by another molecule or compound, such as a carbohydrate, which mimics the natural conformation and function of the amino acid, amino acids or peptide; or a replacement with another molecule or compound, such as a carbohydrate or nucleic acid monomer, that mimics the natural conformation, charge distribution, and function of the parent peptide. Peptides can also be modified by acetylation or amidation.

Synthesis of inhibitory peptide derivatives may be based on known methods for peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, one skilled in the art can use an appropriate computer program to determine the conformation of the peptide of interest. Once the conformation of a peptide disclosed herein has been determined, one skilled in the art, using modeling techniques, can determine the type of substitutions required at a given site to produce a derivative that retains the basic conformation and charge distribution of the parent peptide, but which may have characteristics missing from the parent peptide or improved compared to the characteristics of the parent peptide. Once candidate molecule derivatives have been identified, these derivatives can be tested using the assays described herein to determine their ability to function as MASP-2 inhibitory agents.

Screening for MASP-2 inhibitory peptides.

Molecular modeling and rational molecular design techniques can be used to generate and screen for peptides that mimic the molecular structure of key MASP-2 binding sites and suppress MASP-2-dependent complement activation. The molecular structures used for modeling include the CDRs of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including the site required for dimerization, the site involved in MBL binding, and the serine active site. proteases as described above. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting can be used to de novo design macromolecular structures such as peptides that bind to a particular molecule. See, for example, Shea, K.J., Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP 2:(5), 1994.

As an illustration, the following is an example of one of the ways to obtain mimetics of MASP-2 binding peptides. Functional monomers of a known MASP-2 binding peptide or binding site of an anti-MASP-2 antibody that inhibit MASP-2 activity (matrix) are polymerized. The matrix is then removed, followed by polymerization of the second class of monomers at the location where the matrix was removed to create a new molecule that exhibits one or more desired properties similar to those of the matrix. In addition to obtaining peptides, other MASP-2 binding molecules that are MASP-2 inhibitory agents, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active substances can be obtained in this way. . This method can be used to create a wide variety of biological mimetics that are more stable than their natural counterparts, since they are obtained by free radical polymerization of functional monomers, resulting in a connection with a framework that is not subject to biodegradation.

Synthesis of peptides.

MASP-2 inhibitory peptides can be prepared by methods well known in the art, such as the solid phase synthesis method first described by Merrifield, J. Amer. Chem. Soc., 85:2149-2154, 1963. The 431A Peptide Synthesizer from Applied Biosystems (Foster City, Calif.) can perform automated synthesis according to the manufacturer's instructions. Other methods are described, for example, in Bodanszky, M. et al., Peptide Synthesis, second edition, John Wiley & Sons, 1976, as well as other works known to those skilled in the art.

The peptides can also be obtained by standard genetic engineering methods known to those skilled in the art. For example, a peptide can be obtained by enzymatic synthesis by introducing a nucleic acid encoding a peptide into an expression vector that expresses DNA and translates this DNA into a peptide in the presence of the necessary amino acids. The peptide is then purified by chromatography or electrophoresis or a carrier protein, which can be fused and subsequently cleaved from the peptide, by inserting into an expression vector a nucleic acid sequence encoding the carrier protein along with the peptide encoding sequence. The fusion protein-peptide can be isolated using chromatography, electrophoresis, or immunological methods (eg, coupling the carrier protein to the resin via an antibody). The peptide can be cleaved off chemically or enzymatically, for example with a hydrolase.

The MASP-2 inhibitory peptides used in the method of the invention can also be produced in recombinant host cells using conventional methods. For expression follow

A nucleic acid molecule encoding a 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 control sequences such as promoters and enhancers, expression vectors may include translational control sequences and a marker gene that are suitable for selecting cells carrying the expression vector.

Nucleic acid molecules encoding a MASP-2 inhibitory peptide can be synthesized by gene machines using protocols such as the phosphoramide method. If an application, such as gene synthesis or a gene fragment, requires the chemical synthesis of double-stranded DNA, then each complementary strand is synthesized separately. Obtaining short genes (from 60 to 80 base pairs) will not cause difficulties and can be performed by synthesizing complementary strands followed by annealing. To obtain longer genes, synthetic genes (double-stranded) are assembled into a modular form from single-stranded fragments from 20 to 100 nucleotides in length. For a review of polynucleotide synthesis, see, for example, 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. Sc., USA, 87:633, 1990.

low molecular weight inhibitors.

In some embodiments, the MASP-2 inhibitory agents are small molecule inhibitors, including natural and synthetic low molecular weight agents such as peptides, peptidomimetic agents, and non-peptide inhibitors (including oligonucleotides and organics). Small molecule MASP-2 inhibitors can be derived from the molecular structure of the variable regions of anti-MASP-2 antibodies.

Small molecular weight inhibitors can also be designed and created based on the crystal structure of MASP-2 using computer simulation programs for drug substances (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's crystal structure coordinates are entered into 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, the crystal structure of an HIV-1 protease inhibitor has been used to identify unique non-peptide ligands that are HIV1 protease inhibitors by assessing the compatibility of substances from the Cambridge Crystallographic database with the enzyme binding site using the DOCK program (Kuntz, L.D. et al., J. Mol. Biol., W:269-288, 1982; DesJarlais, R.L et al., PNAS, 87:6644-6648, 1990).

A list of small molecules identified by computer programs as potential inhibitors of MASP-2 are screened in a MASP-2 binding assay such as described in Example 10. Those small molecules that were determined to bind to MASP-2 then assessed in a functional analysis, such as described in example 2, to determine their ability to suppress MASP-2-dependent complement activation.

Soluble MASP-2 receptors.

It is believed that other suitable inhibitory MASP-2 agents include soluble MASP-2 receptors, which can be obtained by methods well known to the person skilled in the art.

MASP-2 expression inhibitors.

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

Antisense RNA and DNA molecules directly block translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA, preventing translation of the MASP-2 protein. The antisense nucleic acid molecule can be designed in various ways and is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or portion thereof) of the MASP-2 cDNA (SEQ ID NO: 4) from its normal transcriptional orientation, allowing for transcription of its complement.

Typically, the antisense nucleic acid molecule is identical to at least a portion of the target gene or genes. However, to suppress the expression of the target gene, the nucleic acid does not have to be absolutely identical to its nucleotide sequence. In general, the shorter the antisense nucleic acid sequence, the higher the homology it should have. The minimum percentage of identity is usually at least about 65%, but

- 44 043102 a higher percentage allows to achieve a more effective suppression of the expression of the endogenous sequence. A percentage identity substantially greater than about 80% is generally preferred, although values ranging from about 95% to complete identity are most preferred.

The antisense nucleic acid molecule need not have an intron or exon of the same structure as the target gene; non-coding segments of the target gene can be as effective in achieving antisense suppression of the expression of the target gene as coding segments. As the antisense nucleic acid molecule, a DNA sequence of 8 or more nucleotides can be used, although a longer sequence is preferred. In the present invention, an exemplary MASP-2 inhibitory agent is a MASP-2 antisense nucleic acid molecule that is at least 90% identical to a complementary MASP-2 cDNA consisting of the nucleic acid sequence shown in SEQ ID NO: 4. 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 antisense oligonucleotides to bind to MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, polygalacturonase and type 2 acetylcholine muscarinic receptor synthesis is inhibited by antisense oligonucleotides targeting the respective mRNA sequences (US Pat. No. 5,739,119 to Cheng and US Pat. No. 5,759,829 to Shewmaker). In addition, an example of antisense suppression has been demonstrated for the core protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA receptor, and human EGF (see, for example, US Pat. No. 5,801,154 to Baracchini ; US Pat. No. 5,789,573 Baker; US Patent 5,718,709 Considine; and US Patent 5,610,288 Reubenstein).

A system has been described to allow one of skill in the art to determine which oligonucleotides may be used for the purposes of the present invention, which involves probing appropriate sites in the target mRNA by RNase H digestion as an indicator of the availability of sequences in transcripts. 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 that are complementary to certain regions of the MASP-2 transcript are added to MASP-2 expressing cell extracts, such as hepatocytes, and hybridized to create sites sensitive to action of RNase H. This method can be combined with sequence selection using a computer program that can predict the selection of optimal sequences for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that would reduce or prevent specific binding to the target mRNA in the host cell. Analysis of these secondary structures and selection of target sites can be performed using the OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN 2.0.5 software (Altschul, S.F. et al., Nucl. Acids Res., 25: 3389-3402, 1997). Antisense compounds directed to a target sequence preferably contain from about 8 nucleotides to about 50 nucleotides. Antisense oligonucleotides containing from about 9 nucleotides to about 35 nucleotides are particularly preferred. The inventors consider all oligonucleotide compositions ranging from 9 to 35 nucleotides (i.e. having a length of 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 nucleotides) as most preferred for methods of the invention based on the use of antisense oligonucleotides. The most preferred target regions of the MASP-2 mRNA are those located in close proximity to the translation initiation codon AUG and sequences that are substantially complementary to the 5' regions of the mRNA, e.g. between the -10 and +10 regions of the nucleotide sequence of the MASP-2 gene. (SEQ ID NO: 4). Examples of inhibitors of MASP-2 expression are presented in table. 4.

- 45 043102

Table 4

Examples of Inhibitors of MASP-2 Expression

SEQ ID NO:30 (nucleotides 22-680 in SEQ ID NO:4) MASP-2 cDNA nucleic acid sequence (SEQ ID NO:4) encoding CUBIEGF SEQ ID NO: 31 5'CGGGCACACCATGAGGCTGCTGA CCCTCCTGGGC3 Nucleotides 12-45 of the SEQ sequence ID NO:4, including site of initiation translation MASP-2 (sense) SEQ ID NO: 32 5'GACATTACCTTCCGCTCCGACTC CAACGAGAAG3' Nucleotides 361-396 of SEQ ID NO:4 encoding the region containing the MASP-2 MBL binding site (sense) SEQ ID NO: 33 5'AGCAGCCCTGAATACCCACGGCC GTATCCCAA3' Nucleotides 610-642 of SEQ ID NO:4 encoding the region that contains the CUBII domain

As mentioned above, as used herein, the term oligonucleotide means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term also encompasses those oligonucleobases that are composed of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) bonds, as well as oligonucleotides having non-naturally occurring modifications. Such modifications allow certain desired properties to be introduced that are not present in naturally occurring oligonucleotides, such as reduced toxicity, improved resistance to nuclease digestion, and increased cellular uptake. In exemplary embodiments, the antisense compounds of the invention differ from native DNA by modifications to the phosphodiester backbone designed to extend the life cycle of an antisense oligonucleotide whose phosphate substituents have been replaced by phosphorothioates. Similarly, one or both ends of the oligonucleotide may be substituted with one or more acridine derivatives that intercalate between adjacent base pairs in the nucleic acid strand.

Another alternative to the use of antisense elements is the use of RNA interference (RNAi). Double-stranded RNAs (dsRNAs) can induce gene silencing in mammals in vivo. The natural function of RNAi and cosuppression appears to be protection of the genome from invasion by mobile genetic elements such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when activated (see, e.g., Jensen, J. et al. , Nat. Genet., 21:209-12, 1999). A double-stranded RNA molecule can be prepared by synthesizing two strands of RNA capable of forming double-stranded RNA molecules each about 19 to 25 (eg, 19-23 nucleotides) in length. For example, the dsRNA molecule used in the methods of the invention may contain an RNA corresponding to the sequence and its complement shown in table. 4. Preferably, at least one RNA strand has a 3' sticky end 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 with a total length of 25 nucleotides or less. Designing siRNA sequences for a given target is well known to those skilled in the art. There are commercial organizations involved in the construction of siRNA sequences that guarantee at least 70% expression knockdown (Qiagen, Valencia, Calif).

The dsRNA can be administered in the form of a pharmaceutical composition by known methods by which the nucleic acid is introduced into the desired target cell. Commonly known gene transfer methods include the calcium phosphate method, the DEAE-dextran method, electroporation, microinjection, and viral methods. Such methods are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

Ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be used to reduce the amount and/or biological activity of MASP-2. Ribozymes are catalytic RNA molecules capable of cleaving nucleic acid molecules with a sequence that is wholly or partially homologous to that of the ribozyme. It is possible to construct ribozyme transgenes encoding RNA ribozymes that form specific pairs with the target RNA and cleave the phosphodiester backbone in a specific site, resulting in functional inactivation of the target RNA. When such a cleavage is carried out, the ribozyme itself does not change and is thus capable of recycling and cleaving other molecules. The incorporation of ribozyme sequences into antisense RNAs results in cleavage activity.

- 46 043102 RNA, thus increasing the activity of antisense constructs.

The ribozymes used in the invention typically contain a hybridizing region of at least nine nucleotides that is complementary to the nucleotide sequence of at least a portion of the target MASP-2 mRNA and a catalytic region that is capable of cleaving the target mRNA (see EPA No. 0321201; WO 88/04300; 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).

Ribozymes can either be targeted directly to cells in the form of RNA oligonucleotides containing ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozyme RNA. Ribozymes can be used and used in much the same way as described for antisense polynucleotides.

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

Various well-known modifications of the DNA molecules can be introduced to improve stability and half-life. Suitable modifications include, without limitation, the addition of flanking ribonucleotide or deoxyribonucleotide sequences to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl in place of phosphodiester bonds within the oligodeoxyribonucleotide backbone.

V. Pharmaceutical compositions and methods of delivery.

Dosing.

In another aspect of the present invention, compositions are provided for suppressing the side effects of MASP-2 dependent complement activation in a subject suffering from a disease or condition as disclosed herein, comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. MASP-2 inhibitory agents may be administered to a subject in need thereof at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2 dependent complement activation. By therapeutically effective dose is meant an amount of a MASP-2 inhibitory agent sufficient to alleviate the symptoms associated with the disease or condition.

The toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models such as the MASP-2-/- mouse model expressing the human MASP-2 transgene described in Example 1. Using such animal models, the values NOAEL (No Observed Side Effect Level) and MED (Minimum Effective Dose) can be determined by standard methods. The dose ratio between the effects of NOAEL and MED is the therapeutic ratio, which is expressed as the NOAEL/MED ratio. MASP-2 inhibitory agents exhibiting high therapeutic ratios or performance are most preferred. Data obtained from studies of cell cultures and animals can be used to determine the range of doses for humans. Doses of the MASP-2 inhibitory agent are preferably in the range of circulating concentrations that include MED with little or no toxicity. The dose may vary within this range depending on the dosage form or the route of administration used.

In some embodiments, the therapeutic efficacy of MASP-2 inhibitory agents for treating, suppressing, alleviating, or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation is determined by one or more of the following:

reduction in renal tissue of one or more markers of inflammation or scarring (eg, TGFe-1, CTFF, IL-6, apoptosis, fibronectin, laminin, collagens, EMT, infiltrating macrophages);

reducing the level of release into the urine or plasma of soluble markers of inflammation and fibrotic kidney disease (for example, by measuring renal excretory function).

For any compound formulation, a therapeutically effective dose can be estimated using animal models. For example, the dose may be adjusted in an animal model to achieve a range of circulating plasma concentrations that includes the MED. Plasma levels of a MASP-2 inhibitory agent can also be measured, for example, using

- 47 043102 HPLC.

In addition to studying toxicity, an effective dose can also be estimated from the amount of MASP-2 protein in a living subject and the binding affinity of the MASP-2 inhibitory agent. Serum levels of MASP-2 in healthy individuals have been shown to be low, in the range of 500 ng/mL, and MASP-2 levels in a given subject can be determined by MASP-2 quantitation methods as described by Moller-Kristensen M. et al., J. Immunol. Methods, 282:159-167, 2003.

Typically, the dose of compositions containing MASP-2 inhibitory agents administered will vary depending on factors such as the subject's age, body weight, height, sex, general health, and medical history. By way of illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered in a dose range of about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg subject's body weight. In some embodiments, the composition comprises a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.

The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the invention in a given subject, as well as appropriate dosages, can be determined by complement analysis well known to one of skill in the art. Complement generates numerous specific products. Over the past ten years, precise and specific assays have been developed that are commercially available for most of these activation products, including small activation fragments C3a, C4a and C5a and large activation fragments iC3b, C4d, Bb and sC5b-9. Most of these assays use monoclonal antibodies that react to novel antigens (neoantigens) located on these fragments, but not to the native proteins from which they are derived, making these assays highly accessible and specific. In most cases, the ELISA method is used, although radioimmunoassay is still sometimes used for C3a and C5a. The latter assay is used to measure the level of both unprocessed fragments and their 'desArg' fragments, which are the main forms found in the bloodstream. Non-processable fragments and C5a _desArg are rapidly cleared via binding to cell surface receptors and are therefore present at very low concentrations, while C3a _des A _rg does not bind to cells and accumulates in plasma. C3a measurement is a sensitive, independent indicator of complement activation. An alternative complement activation pathway can be determined by measuring the Bb fragment. The detection of soluble activation products of the membrane attack pathway, sC5b-9, is evidence that complement is fully activated. Since both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of the two complement pathways generated the activation products.

Suppression of MASP-2 dependent complement activation is characterized by at least one of the following changes in a component of the complement system that results from administration of an inhibitory MASP-2 agent in accordance with the methods of the invention: suppression of the generation or production of products of the MASP-2 dependent activation system complement: C4b, C3a, C5a and/or C5b-9 (measured, for example, as described in example 2), reduction in the level of splitting of C4 and deposition of C4b (measured, for example, as described in example 10) or decrease in the level of splitting of C3 and deposition C3b (measured, for example, as described in example 10).

additional agents.

In some embodiments, methods for preventing, treating, reversing, and/or suppressing fibrosis and or inflammation comprise administering a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) as part of a therapeutic regimen with one or more other drugs, biologicals, or therapeutic agents. treatments suitable for suppressing fibrosis and/or inflammation. In some embodiments, the implementation of additional drug, biological agent or therapeutic method of treatment is suitable for specific symptoms associated with a disease or disorder caused or complicated by fibrosis and/or inflammation. As an example, MASP-2 inhibitory antibodies can be administered as part of a therapeutic regimen along with one or more immunosuppressants such as methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil. As another example, MASP-2 inhibitory antibodies can be administered as part of a therapeutic regimen along with one or more blood flow enhancing agents (eg, nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As another example, MASP-2 inhibitory antibodies can be administered as part of a therapeutic regimen along with one or more fibrosis reducing agents such as d-penicillamine, colchicine, PUVA, relaxin, cyclosporine, TGF beta blockers, and/or p38 MAPK blockers. As a further example, MASP-2 inhibitory antibodies can be administered as part of a therapeutic regimen along with steroids or bronchodilators.

Compositions and methods using MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) may optionally include one or more additional therapeutic

- 48 043102 agents that are capable of enhancing the activity of an inhibitory MASP-2 agent or that provide related therapeutic functions, providing an additional or synergistic effect. For example, in the context of treating a subject suffering from a disease or disorder caused or complicated by fibrosis and/or inflammation, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with one or more additional antifibrotic agents and/or one or more more anti-inflammatory agents and/or immunosuppressants.

MASP-2 inhibitory agents (eg, MASP-2 inhibitory antibodies) can be used in combination with other therapeutic agents such as general immunosuppressants such as corticosteroids, immunosuppressants or cytotoxic agents and/or antifibrotic agents.

Pharmaceutical carriers and means of delivery.

In general, the MASP-2 inhibitory agent compositions of the present invention, when combined with any other selected therapeutic agent, are contained in a suitable pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and is chosen such that adverse effects on the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agent included in the combination) can be avoided. Exemplary pharmaceutically acceptable peptide carriers are described in US Pat. No. 5,211,657 to Yamada. Ahtu-MASP-2 antibodies and inhibitory peptides used in the present invention can be formulated into solid, semi-solid, gel, liquid and gas dosage forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalations and injections. for oral, parenteral or surgical administration. The present invention also provides for topical administration of the compositions by coating medical instruments and the like.

Suitable vehicles for parenteral administration by injection, infusion or irrigation and topical delivery are distilled water, phosphate buffered saline, Ringer's solution, normal or lactated, D-glucose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils can be used as a solvent or suspending medium. Any biocompatible oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectable preparations. The carrier and agent may be mixed as liquid solutions, suspensions, polymerizable or non-polymerizable gels, pastes or balms.

The carriers may also contain a delivery device to slow (ie, prolong, delay or control) delivery of the agent(s) or to enhance the delivery, absorption, stabilization or pharmacokinetic parameters of the therapeutic agent(s). Such a delivery vehicle may include, without limitation, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organics, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes described in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes described in US Patent Application Publication 2002/0019369 A1. Such hydrogels may be administered by local injection at the site of intended action or subcutaneously or intramuscularly to provide sustained release of the agent.

In the case of intra-articular delivery, the MASP-2 inhibitory agent can be delivered in the above-described injectable liquid or gel carriers, the above-described sustained release injectable delivery vehicles, or carriers based on hyaluronic acid or its derivatives.

In the case of oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be delivered in inert vehicles or solvents such as sucrose, corn starch or cellulose.

In the case of topical administration, the MASP-2 inhibitory agent can be delivered as an ointment, lotion, cream, gel, drops, suppository, spray, liquid or powder, or in gel or microcapsule delivery systems via a transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, are under development and can be appropriately adapted to deliver the formulation of the present invention via an aerosol, inhaler, or nebulizer delivery vehicle, respectively.

In the case of intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriate sterile delivery systems (eg, liquids, gels, suspensions, etc.) may be used.

The compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, solvents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents.

- 49 043102 substances (for oral administration).

Pharmaceutical carriers for antibodies and peptides.

More specifically, with respect to anti-MASP-2 antibodies and inhibitory peptides, the exemplary formulations may be administered parenterally as dosed injections of a solution or suspension of the compound in a physiologically acceptable vehicle with a pharmaceutical carrier, which may be a sterile liquid such as water, oil , saline, glycerin or ethanol. In compositions containing anti-MASP-2 antibodies and inhibitory peptides, adjuvants such as wetting or emulsifying agents, surfactants, pH adjusting agents, and the like may additionally be present. Additional components of pharmaceutical compositions include a hydrocarbon mixture (such as animal, vegetable or synthetic origin), for example, soybean oil and mineral oil. For injectable solutions, the preferred liquid carriers are generally glycols such as propylene glycol and polyethylene glycol.

Ahtu-MASP-2 antibodies and inhibitory peptides can also be administered in the form of injections of depot agents or an implantable preparation that can be formulated to provide sustained or pulsatile release of the active agents.

Pharmaceutically acceptable carriers for expression inhibitors.

With respect to expression inhibitors used in the methods of the invention, compositions are provided comprising an expression inhibitor as described above and a pharmaceutically acceptable carrier or diluent. The compositions may additionally contain a colloidal dispersion system.

Pharmaceutical compositions containing expression inhibitors may include, without exception, formulations containing solutions, emulsions and liposomes. Such compositions can be prepared from various components, including, without limitation, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. 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 dextrin, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of appropriate solvents.

In some embodiments, the compositions may be formulated and formulated as emulsions, which are typically heterogeneous systems of one liquid substance dispersed in another as microdroplets (see Idson, in Pharmaceutical Dosage Forms, vol. 1, Rieger and Banker (eds.) , Marcek Dekker, Inc., N.Y., 1988). Examples of natural emulsifying agents used for emulsion formulations are gum arabic, beeswax, lanolin, lecithin, and phosphatides.

In one embodiment, compositions containing nucleic acids may be formulated as microemulsions. A microemulsion, as described herein, is a water, oil, and amphiphile system that is a single optically homogeneous and thermodynamically stable liquid composition (see Rosoff in Pharmaceutical Dosage Forms, vol. 1). The method of the invention may also include the use of liposomes to transport and deliver antisense oligonucleotides to the desired site.

Pharmaceutical compositions and topical expression inhibitor formulations may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, such as those based on water, powders or oils, thickeners, and the like, may also be used.

Methods of administration.

Pharmaceutical compositions containing MASP-2 inhibitory agents may be administered in a variety of ways, depending on which route of administration, topical or systemic, is more appropriate for the disease being treated. Further, the compositions of the present invention can be delivered by coating or incorporating the compositions into the surface of an implantable medical device or into the device itself.

System delivery.

As used herein, the terms systemic delivery or systemic administration include, without limitation, oral and parenteral routes of administration, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal , vaginal, and other routes of administration that provide effective dispersion of the delivered agent at one or more sites of intended therapeutic effect. Preferred methods of systemic delivery of the present compositions include intravenous, intramuscular, subcutaneous and inhalation. It will be understood that the precise mode of systemic administration of selected agents used in particular compositions of the present invention can be determined by considering that agent's susceptibility to metabolic transformation in a manner associated with that mode of administration. For example, the most suitable route of administration for peptidergic agents is any other than oral.

Antibodies and polypeptides that inhibit MASP-2 can be delivered to a subject in need thereof by any suitable means. Methods for delivering MASP-2 antibodies and polypeptides include oral, pulmonary, parenteral administration (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injections), inhalation (e.g., fine powder), transdermal, nasal, vaginal, rectal, or subcutaneous administration, and may be formulated into dosage forms suitable for each of the routes of administration.

As an illustrative example, MASP-2 inhibitory antibodies and peptides can be administered to a living body by application to a mucosal surface capable of absorbing the polypeptides, such as the nasal, gastrointestinal, and rectal mucosa. The polypeptides are usually applied to an absorbent mucosa in combination with a penetration enhancing agent (see, for example, Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys., 5:69, 1988; Lee, V.H.L., J. Controlled Release, 13:213, 1990; Lee, V.H.L., ed., 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, a steroidal surfactant that is similar in structure to bile salts and is used as a penetration enhancing agent in nasal delivery. (Lee, W.A., Biopharm., 22, Nov./Dec. 1990.)

Antibodies and polypeptides that inhibit MASP-2 may be administered in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body, and thus prolong their half-life (Fuertges, F. et al., J. Controlled Release, 11:139 , 1990). Many polymeric protein delivery systems have been described (Bae, Y. H. et al., J. Controlled Releas, e 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; Makino, K., J. Controlled Release, 12:235, 1990; Takakura, Y et al., J. Pharm Sci. 78:117, 1989; Takakura, Y. et al., J. Pharm. Sci., 78:219, 1989).

Recently, liposomes have been developed with improved serum stability and extended half-life (see, for example, US Pat. No. 5,741,516 to Webb). In addition, various methods for the production of liposomes and liposome-like preparations as potential drug carriers have been considered (see, for example, US Pat. No. 5,567,434 to Szoka; US Pat. No. 5,552,157 to Yagi; US Pat. USA 5795587, Gao).

For transdermal application, MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable ingredients such as carriers and/or adjuvants. Such ingredients are used regardless of their nature, except that they must be pharmaceutically acceptable for the intended administration and must not reduce the effect of the active ingredients of the composition. Examples of suitable bases include creams, ointments, gels or suspensions, with or without purified collagen. MASP-2 inhibitory antibodies and polypeptides can also be impregnated into transdermal patches, dressings and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be administered systemically at intervals necessary to maintain the desired level of therapeutic effect. For example, formulations may be administered by subcutaneous injection every two to four weeks, or at less frequent intervals. The dosage regimen is determined by the attending physician, taking into account various factors that may affect the effect of the combination of agents. These factors include the degree of progression of the disease being treated, the patient's age, sex and body weight, and other clinical factors. The dose of each individual agent will vary depending on the function of the MASP-2 inhibitory agent included in the composition, as well as the presence and nature of any drug delivery vehicle (eg, sustained release delivery vehicle). In addition, the dosage can be adjusted based on the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

Local delivery.

As used herein, the term topical encompasses the application of a drug to or around the area of intended local action and may include, for example, topical delivery to the skin or other affected tissue, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavitary, intracranial, or intravesical administration, placement or irrigation. Topical administration may be preferred in terms of lower doses, prevention of systemic side effects, and more precise control of delivery time and concentration of active agents at the site of local delivery. Local administration provides known concentrations in the target area, regardless of

- 51 043102 individual differences between patients in terms of metabolism, circulation, etc.

Improved dosing control is also provided by direct delivery.

Local delivery of a MASP-2 inhibitory agent can be achieved in the context of surgical treatments for diseases or disorders caused or complicated by fibrosis and/or inflammation, for example during procedures such as surgery.

Treatment regimens.

For prophylactic purposes, pharmaceutical compositions containing a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) are administered to a subject susceptible to or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation, in an amount sufficient to suppress fibrosis and /or inflammation, thus eliminating or reducing the risk of developing symptoms of this condition. In some embodiments, the pharmaceutical compositions are administered to a subject suspected of having, or already suffering from, a disease or disorder caused by or complicated by fibrosis and/or inflammation, in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of that condition. . For both prophylactic and therapeutic purposes, compositions containing an inhibitory MASP-2 agent may be administered in multiple doses until a sufficient therapeutic result is achieved. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or a limited series of administrations for the treatment of an acute condition associated with fibrosis and/or inflammation. Alternatively, the composition may be administered at regular intervals over an extended period of time to treat a chronic condition associated with fibrosis and/or inflammation.

For both prophylactic and therapeutic purposes, compositions containing an inhibitory MASP-2 agent may be administered in multiple doses until a sufficient therapeutic result is achieved. In one embodiment, the MASP-2 inhibitory agent comprises a MASP-2 inhibitory antibody, which can preferably be administered to an adult patient (e.g., an adult patient with an average body weight of 70 kg) at a dose ranging from 0.1 to 10,000 mg, more preferably 1.0 to 5000 mg, more preferably 10.0 to 2000 mg, more preferably 10.0 to 1000 mg, and even more preferably 50.0 to 500 mg. For pediatric patients, the dose may be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or a limited series of administrations for the treatment of a subject suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation. Alternatively, the composition may be administered at regular intervals, such as daily, twice a week, once a week, every other week, once a month, twice a month for an extended period of time to treat a subject suffering from or at risk of developing a disease or disorder, caused or complicated by fibrosis and/or inflammation.

For both prophylactic and therapeutic purposes, compositions containing an inhibitory MASP-2 agent may be administered in multiple doses until a sufficient therapeutic result is achieved.

In some embodiments, a subject is identified as at risk of developing a disease or disorder caused or complicated by fibrosis or inflammation by determining that the subject has one or more symptoms of impaired renal function, as assessed by, for example, measuring serum creatinine, serum creatinine clearance , urinary nitrogen, urinary protein, and/or by measuring one or more biomarkers associated with kidney disease or injury.

Methods for assessing kidney function are well known in the art and include, but are not limited to, measurements of systemic blood pressure and glomerular capillary pressure, proteinuria (e.g., albuminuria), microscopic and gross hematuria, serum creatinine (e.g., according to one of the human kidney function assessment formulas). , a creatinine level of 2.0 mg/dl corresponds to 50% of normal renal function, and 4.0 mg/dl corresponds to 25%), a decrease in glomerular filtration rate (eg, creatinine clearance rate) and the degree of tubular damage. For example, evaluation of kidney function may include evaluation of at least one kidney function using biological and/or physiological parameters such as serum creatinine, creatinine clearance rate, 24-hour protein secretion, glomerular filtration rate, creatinine to albumin ratio in urine, the rate of excretion of albumin (for example, determining the degree of renal fibrosis by measuring the deposition of collagen and / or fibronectin).

VI. Examples.

The following examples are only illustrative of the best way to practice the present invention and should not be construed as limiting the present invention. All literature references mentioned in this application are incorporated herein by reference.

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Example 1

This example describes the production of a mouse line deficient in MASP-2 (MASP-2-/-) but complete in MAp19 (MAp19+/+).

Materials and methods. The target vector pKO-NTKV 1901 was designed to destroy three exons encoding the C-terminal end of mouse MASP-2, including the exon that encodes the serine protease domain, as shown in FIG. 3. PKO-NTKV 1901 was used to transfect the mouse ES cell line E14.1a (SV129 Ola). Clones resistant to neomycin and sensitive to thymidine kinase were selected. 600 ES clones were screened and four different clones containing the desired selective target and recombinant events were identified and verified by Southern blot analysis as shown in FIG. 3. Chimeras were generated from these four positive clones by embryo transfer. The chimeras were then backcrossed to the C57/BL6 background phenotype to produce transgenic males. The transgenic males were then crossed with females to produce F1 with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. Heterozygous mice were crossed with each other to produce homozygous offspring deficient in MASP-2, resulting in a ratio between heterozygous mice and wild-type mice of 1:2:1, respectively.

Results and phenotype. The resulting homozygous MASP-2-/- deficient mice that were viable and fertile were tested for MASP-2 deficiency using Southern blot analysis to confirm the correct target event, using Northern blot analysis to confirm the absence of MASP-2 mRNA. 2 and using Western blot analysis to confirm the absence of MASP-2 protein (data not shown). The presence of MAp19 mRNA and the absence of MASP-2 mRNA were further confirmed by time resolved PCR-PCR on a LightCycler device. As expected, MASP-2 −/− mice continued to express mRNA and MAp19, MASP-1 and MASP-3 proteins (data not shown). MASP-2-/- mice were analyzed for the presence and excess mRNA of properdin, factor B, factor D, C4, C2, and C3 by LightCycler analysis and were found to be identical to levels found in control wild-type littermates (data not shown). The plasma of homozygous MASP-2 -/- mice was completely deficient in lectin-mediated complement activation, as described in Example 2 below.

Generation of MASP-2-/- lines on the pure background genotype C57BL6. MASP-2-/- mice were backcrossed to pure C57BL6 for nine generations prior to using the MASP-2-/- line as an experimental animal model.

A transgenic mouse line which is a mouse MASP-2-/-, MAp19+/+ line and which expresses the human MASP-2 transgene (mouse MASP-2 knockout and human MASP-2 knockin) was generated as follows:

Materials and methods. A minigene encoding human MASP-2, named minihMASP-2 (SEQ ID NO: 49), was created as shown in FIG. 4, which includes the promoter region of the human MASP-2 gene, including the first three exons (exon 1 to exon 3), followed by a cDNA sequence that is a coding sequence of 8 consecutive exons, encoding a full-length MASP-2 protein, driven by an endogenous promoter. The mini-hMASP-2 construct was introduced into fertilized MASP-2-/- eggs to replace the deficient mouse MASP-2 gene with the transgenic expressed human MASP-2.

Example 2

This example shows that MASP-2 is required for complement activation via the lectin pathway.

Materials and methods.

Analysis of C4 cleavage specific to the lectin pathway. C4 cleavage assay is described in Petersen et al., J. Immunol. Methods, 257:107 (2001), which measured lectin pathway activation initiated by lipoteichoic acid (LTA) from S. aureus, which binds to L-ficolin. The assay described by Petersen et al., (2001) was adapted to measure lectin pathway activation by MBL by coating the plate with LPS and mannan or zymosan prior to addition of serum derived from MASP-2-/- mice, as described below. The assay was also modified to eliminate the possibility of classical C4 cleavage. This was achieved by using a sample dilution buffer containing 1 M NaCl, which provided a high binding affinity for the recognition components of the lectin pathway to their ligands, but prevented activation of endogenous C4, thus precluding involvement of the classical pathway by dissociation of the C1 complex. Briefly, in a modified assay, serum samples (diluted with high salt buffer (1 M NaCl)) were added to ligand coated plates followed by the addition of a constant amount of purified C4 in physiological salt buffer. Bound recognition complexes containing MASP-2 cleave C4 leading to the deposition of C4b.

Methods of analysis.

1) Nunc Maxisorb microtiter plates (MaxiSorb®, Nunc, cat. no. 442404, Fisher Scientific) were coated with 1 µg/ml mannan (M7504 Sigma) or any other ligand (such as trans-

- 53 043102 numerals below) diluted in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

The following reagents were used in the assay:

a) mannan (1 µg per well of mannan (M7504 Sigma) in 100 µl coating buffer);

b) zymosan (1 μg per well of zymosan (Sigma) in 100 μl coating buffer);

c) LTA (1 µg/well in 100 µl coating buffer or 2 µg/well in 20 µl methanol);

d) 1 μg H-ficolin-specific Mab 4H5 in coating buffer;

e) PSA from Aerococcus viridans (2 μg per well in 100 μl coating buffer);

f) 100 µl per well of S. aureus DSM20233 fixed in formalin (OD ₅₅₀ = 0.5) in coating buffer.

2) The plate was incubated overnight at 4°C.

3) After overnight incubation, the remaining protein binding sites were saturated by incubating the plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris-Cl, 140 mM NaCl, 1.5 mM NaN ₃ , pH 7.4) for 1-3 h and then the plates were washed three times with a mixture of TBS/Tween/Ca ²⁺ (TBS with 0.05% Tween 20 and 5 mm CaCl ₂ , 1 mm MgCl ₂ , pH 7, 4).

4) Serum samples to be tested were diluted in MBL binding buffer (1 M NaCl) and the diluted samples were added to the plates and incubated overnight at 4°C. Wells to which only buffer was added were used as negative controls.

5) After overnight incubation at 4°C, the plates were washed three times with a mixture of TBS/Tween/Ca ²⁺ . Then human C4 (1 μg/ml diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4) 100 μl per well) was added to the plate and incubated for 90 min. at 37°C. The plates were then washed three times again with TBS/Tween/Ca ²⁺ .

6) C4b deposition was determined with alkaline phosphatase-conjugated chicken anti-human C4c (diluted 1:1000 in TBS/Tween/Ca ²⁺ ), which was added to the plates and incubated for 90 min at room temperature. The plates were then washed three times again with TBS/Tween/Ca ²⁺ .

7) Alkaline phosphatase was detected by adding 100 μl of p-nitrophenyl phosphate substrate solution, incubating at room temperature for 20 minutes and reading the absorbance OD ₄₀₅ on a microtiter plate reader.

Results.

In FIG. 5A, 5B show C4b deposition on mannan (FIG. 5A) and zymosan (FIG. 5B) in diluted sera derived from MASP-2+/+ (crosses), MASP-2+/- (closed rings) and MASP-2 -/- (closed triangles) mice. In FIG. 5C shows the relative activity of C4 convertase on zymosan (white bars) or mannan (dark bars) coated plates for MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to mice. wild-type (n=5) as measured by C4b deposition normalized to serum from wild-type mice. Error bars represent the standard deviation. As shown in FIG. 5A-5C, in mannan- and zymosan-coated plates, plasma derived from MASP-2 −/− mice is completely deficient in lectin-mediated complement activation. These results clearly demonstrate that MASP-2, and not MASP-1 or MASP-3, is the effector component of the lectin pathway.

Recombinant MASP-2 restores lectin-mediated C4 activation in serum derived from MASP-2 -/- mice.

To establish that the absence of MASP-2 in MASP-2 −/− mice is the direct cause of the loss of lectin pathway dependent C4 activation, the effect of adding recombinant MASP-2 protein to serum samples was assessed using the C4 cleavage assay described above. Recombinant proteins, functional mouse MASP-2 and catalytically inactive mouse MASP-2A (in which a serine residue in the active site of the serine protease domain is replaced by an alanine residue) were prepared and purified as described in Example 3 below. Pooled serum obtained from four MASPs -2-/- mice were preincubated with increased concentrations of recombinant mouse MASP-2 or inactive recombinant mouse MASP-2A, and C4 convertase activity was assessed as described above.

Results. As shown in FIG. 6, addition of functional mouse recombinant MASP-2 protein (shown by white triangles) to MASP-2-/mice-derived serum restored lectin pathway-dependent C4 activation in a protein concentration-dependent manner, with catalytically inactive mouse MASP protein -2A (shown by asterisks) did not restore C4 activation. The results shown in FIG. 6 was normalized to the C4 activation seen in pooled wild-type mouse sera (indicated by the dotted line).

Example 3

This example shows recombinant expression and production of recombinant full length human, rat and mouse MASP-2 proteins, MASP-2 derived polypeptides and catalytically inactive MASP-2 mutants.

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Expression of full length human, rat and mouse MASP-2.

The full-length human MASP-2 cDNA sequence (SEQ ID NO: 4) was also 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, 185:531-66 (1991)). Full-length mouse cDNA (SEQ ID NO: 50) and rat MASP-2 cDNA (SEQ ID NO: 53) were subcloned into the pED expression vector. MASP-2 expression vectors were then transfected into adherent Chinese Hamster Ovary cell lines DXB1 following the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs developed very slowly, which can be explained by the cytotoxicity of the encoded protease.

In another approach, a minigene construct (SEQ ID NO:49) containing human MASP-2 cDNA driven by an endogenous promoter was transiently transfected in Chinese hamster ovary (CHO) cells. The human MASP-2 protein secreted into the culture medium was isolated according to the scheme described below.

Expression of full length catalytically inactive MASP-2.

Rationale. MASP-2 was activated by autocatalytic cleavage after binding of MBL recognition subcomponents or ficolins (either L-ficolin, H-ficolin or M-ficolin) to their respective carbohydrate molecules. Autocatalytic cleavage leading to MASP-2 activation often occurs during the procedure for isolating MASP-2 from serum or during purification after recombinant expression. To obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, designated MASP-2A, was generated by replacing a serine residue that is present in the catalytic triad of the protease domain with an alanine residue in rats (SEQ ID NO: 55 Ser617 with Ala617); in mice (SEQ ID NO: 52 Ser617 on Ala617); or in humans (SEQ ID NO: 6 Ser618 on Ala618).

To obtain catalytically inactive human and mouse MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides shown in Table 1. 5. Oligonucleotides shown in table. 5 was created by annealing with a region of human and mouse cDNA encoding an enzymatically active serine, and the oligonucleotide contained a mismatch to change the serine codon to an alanine codon. For example, PCR oligonucleotides SEQ ID NOS: 56-59 were used in combination with human cDNA MASP-2 (SEQ ID NO: 4) to amplify the region from start codon to enzymatically active serine and from serine to stop codon to obtain a complete open reads from a mutated MASP-2A containing the Ser618/Ala618 mutation. PCR products were purified after agarose gel electrophoresis and banding, and single adenosine overlaps were obtained using a standard extension procedure. Adenosine-supplemented MASP-2A was then cloned into the pGEM-T Easy vector and transformed into E. coli.

The catalytically inactive rat MASP-2A protein was prepared by subjecting SEQ ID NO: 64 and SEQ ID NO: 65 to kinase and annealing by combining the two oligonucleotides in equal molar volumes, heating at 100° C. for 2 min, and cooling slowly at room temperature. temperature. The annealed fragment contained Pst1 and Xba1 compatible ends and was inserted in place of the wild-type rat MASP-2 cDNA fragment (SEQ ID NO: 53) Pst1-Xba1 to give rat MASP-2A.

'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO: 64) 5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO: 65)

Human, mouse and rat MASP-2A were then each subcloned into pED or pCI-Neo mammalian expression vectors and transfected into the Chinese Hamster Ovary DXB1 cell line as described below.

In another approach, a catalytically inactive form of MASP-2 was prepared by the method described in Chen et al., J. Biol. Chem., 276(28), 25894-25902, 2001. Briefly, a plasmid containing the full-length human MASP-2 cDNA (described in Thiel et al. Nature 386:506, 1997) was cut with Xho1 and EcoR1 and MASP-2 cDNA ( given in the present description in SEQ ID NO: 4) were cloned at the appropriate restriction sites in the baculovirus transfer vector pFastBad (Life Technologies, NY). Then, Ser618 in the active site of the MASP-2 serine protease was replaced by Ala618 by replacing the double-stranded oligonucleotides encoding the peptide region from amino acids 610 to 625 (SEQ ID NO:13) with the natural region from amino acids 610 to 625 to obtain a full-length MASP-2 polypeptide with an inactive protease. domain.

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

To allow secretion of different MASP-2 domains, the following constructs were made using the MASP-2 signal peptide (residues 1-15 in SEQ ID NO: 5). A construct expressing the CUBI domain of human MASP-2 (SEQ ID NO: 8) was obtained by PCR amplification of the region encoding residues 1-121 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBI). A construct expressing the CUBIEGF domain of human MASP-2 (SEQ ID NO: 9) was generated

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PCR amplification of the region encoding residues 1-166 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGF). A construct expressing the CUBIEGFCUBII domain of human MASP-2 (SEQ ID NO: 10) was obtained by PCR amplification of the region encoding residues 1-293 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGFCUBII). The above domains were amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, according to established PCR methods. The 5' primer sequence of the sense primer ( ^5' -CGGGATCCATGAGGCTGCTGACCCCTC-3' SEQ ID NO: 34) was introduced with a BamHI restriction site (underlined) at the 5'end of the PCR product. Antisense primers for each MASP-2 domain shown in Table 1 below. 5 was generated by introducing a stop codon (bold) followed by an EcoRI site (underlined) at the end of each PCR product. After amplification, the DNA fragments were treated with BamHI and EcoRI restriction enzymes and cloned into the corresponding regions of the pFastBacl vector. The resulting constructs were characterized by restriction mapping and confirmed by dsDNA sequencing.

Table 5

PCR primers for MASP-2

MASP-2 domain 5' PCR primer 3' PCR primer SEQ ID NO: 8 CUBI (aa 1-121 in SEQ ID NO:6) 5'CGGGATCCATGAG 5'GGAATTCCTAGGCTGCA GCTGCTGACCCTC-3' (SEQ ID NO:34) TA (SEQ ID NO:35) SEQ ID NO: 9 CUBIEGF (aa 1-166 in SEQ ID NO:6) 5'CGGGATCCATGAG 5'GGAATTCCTACAGGGCG GCTGCTGACCCTC-3' (SEQ ID NO:34) CT-3' (SEQ ID NO:36) SEQ ID NO: 10 CUBIEGFCUBII (aa 1- 293 in SEQ ID NO:6) 5'CGGGATCCATGAG 5'GGAATTCCTAGTAGTGG GCTGCTGACCCTC-3' (SEQ ID NO:34) AT 3' (SEQ ID NO:37) SEQ ID NO: 4 Human MASP-2 5'ATGAGGCTGCTGA CCCTCCTGGGCCTTC-3' (SEQ ID NO: 56) hMAS P-2_straight 5'TTAAAATCACTAATTAT GTTCTCGATC-3' (SEQ ID NO: 59) hMASP2_reverse SEQ ID NO: 4 human cDNA MASP-2 5'CAGAGGTGACGCA GGAGGGGCAC 3' (SEQ ID NO: 58) hMASP2_a1a_straight 5'GTGCCCCTCCTGCGTCA CCTCTG-3’ (SEQ ID NO: 57) hMASP- 2_ala_reverse SEQ ID NO: 50 mouse MASP-2 cDNA 5'ATGAGGCTACTCA TCTTCCTGG3' (SEQ ID NO: 60) mMASP- 2_straight 5'TTAGAAATTACTTATTAT GTTCTCAATCC3' (SEQ ID NO: 63) mMASP2_reverse SEQ ID NO: 50 mouse MASP-2 cDNA 5'CCCCCCCTGCGTC ACCTCTGCAG3' (SEQ ID NO: 62) mMASP2_a1a_straight 5'CTGCAGAGGTGACGCAG GGGGGG 3' (SEQ ID NO: 61) mMASP- 2_ala_reverse

Eukaryotic expression of recombinant MASP-2 and production of enzymatically inactive murine, rat and human MASP-2A proteins.

The above MASP-2 and MASP-2A expression constructs were transfected into DXB1 cells using a standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was obtained in a serum-free nutrient medium to avoid contamination of preparations with other serum proteins. A day later, confluent cells were collected from nutrient media (in total, the process was carried out four times). The average level of recombinant MASP-2A for each of the three species was approximately 1.5 mg/l culture medium.

Purification of the MASP-2A protein. MASP-2A (the Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns. This strategy allows fast cleaning without the use of external markers. MASP-2A (100-200 ml culture medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl ₂ ) was loaded onto an MBP-agarose (4 ml) column for affinity chromatography pre-equilibrated with 10 ml loading buffer After washing with an additional 10 ml loading buffer, the protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5 containing 1.25 M NaCl and 10 mM EDTA.

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Fractions containing MASP-2A were identified by SDS polyacrylamide gel electrophoresis. If necessary, MASP-2A was further purified by ion exchange chromatography on a MonoQ column (MR 5/5). The protein was dialyzed with 50 mM Tris-Cl pH 7.5 containing 50 mM

NaCl, and loaded onto a column equilibrated with the same buffer. After washing, bound MASP-2A was eluted with 10 ml of 0.05-1 M NaCl gradient solution.

Results. The yield of MASP-2A protein was 0.25-0.5 mg per 200 ml of medium. The molecular weight determined by the MALDI-MS method was 77.5 kDa, which exceeded the calculated value for the unmodified polypeptide (73.5 kDa) due to glycosylation. Attachment of glycans to each of the N-glycosylation sites explains the observed mass. MASP-2A migrates migrated as a single band in the SDS polyacrylamide gels, indicating that it was not proteolytically degraded during biosynthesis. The weight average molecular weight determined by equilibrium ultracentrifugation is consistent with the calculated value for glycosylated polypeptide homodimers.

Obtaining recombinant human MASP-2 polypeptides.

Another method for producing recombinant derivatives of MASP-2 and MASP-2A polypeptides is described in Thielens, N.M. et al., J. Immunol., 166:5068-5077, 2001. Briefly, Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) are grown and maintained in Sf90011 serum-free nutrient medium (Life Technologies) containing 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). Trichoplusia ni insect cells (High Five) obtained from Jadwiga Chroboczek, Institut de Biologic Structurale, Grenoble, France) are maintained in TC100 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 IU/ ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses are obtained using the Bac-to-Bac system (Life Technologies). Bacmid DNA was purified with a Qiagen midiprep purification system (Qiagen) and used to transfect insect Sf9 cells using selectin in Sf900 II SFM medium (Life Technologies) according to the manufacturer's protocol. Recombinant viral particles are harvested after 4 days, titrated by the viral plaque method and amplified as described in King and Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, p.111-114, 1992.

High Five cells (1.75x107 cells/175-cm ² tissue culture in flask) were infected with recombinant viruses containing MASP-2 polypeptides by multiple infection, 2 times, in Sf900 II SFM medium at 28°C for 96 hours. Supernatants collected by centrifugation and diisopropyl phosphorofluoridate was added to obtain a final concentration of 1 mm.

MASP-2 polypeptides were secreted in cell culture into a nutrient medium. Culture supernatants were dialyzed against 50 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 8.1 and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm ) balanced with the same buffer. Elution was performed using a 1.2 L linear gradient in 350 mM NaCl in the same buffer. Fractions containing recombinant MASP-2 polypeptides were identified by Western blot analysis, precipitated by adding (NH ₄ ) ₂ SO ₄ to 60% (by weight) and left overnight at 4°C. The pellets were resuspended in 145 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 7.4 and applied to a TSK G3000 SWG (7.5x600 mm) column (Tosohaas, Montgomeryville, PA) equilibrated with the same buffer. The purified polypeptides were then concentrated to 0.3 mg/ml by ultrafiltration in Microsep microconcentrators (threshold MW=10,000) (Fiitron, Karlstein, Germany).

Example 4

This example describes the production of polyclonal antibodies to MASP-2 polypeptides.

Materials and methods.

MASP-2 antigens. A polyclonal anti-human MASP-2 antiserum was prepared by immunizing rabbits with the following isolated MASP-2 polypeptides:

human MASP-2 (SEQ ID NO: 6) isolated from serum;

recombinant human MASP-2 (SEQ ID NO: 6), MASP-2A containing an inactive protease domain (SEQ ID NO: 13) 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.

polyclonal antibodies. Six-week-old rabbits primed with BCG (bacille Calmette-Guerin vaccine) were immunized by injecting 100 μg of the MASP-2 polypeptide at a concentration of 100 μg/ml in saline. Injections were made every 4 weeks with antibody titer monitored by ELISA as described in Example 5. Culture supernatants were collected for antibody purification by protein A affinity chromatography.

Example 5

This example describes a method for producing mouse monoclonal antibodies against rat or human MASP-2 polypeptides.

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Materials and methods.

Male A/J mice (Harlan, Houston, Tex.) aged 8-12 weeks were injected subcutaneously with 100 μg of human or rat rMASP-2 or rMASP-2A polypeptides (prepared as described in Example 3) in Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.) in 200 µl phosphate buffered saline (PBS), pH 7.4. Mice received two subcutaneous injections of 50 μg of human or rat rMASP-2 or rMASP-2A polypeptide in incomplete Freund's adjuvant at two-week intervals. At week 4, the mice received an injection of 50 μg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and 4 days later the mice were used for fusion.

For each fusion, single cell suspensions were prepared from immunized mouse spleens and used to fuse with Sp2/0 myeloma cells. Fusion of 5x108 Sp2/0 cells and 5x108 spleen cells was performed in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.). Cells were then adjusted to 1.5x105 spleen cells in 200 µl suspension in Iscove medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine. Two hundred microliters of cell suspension was added to each well of about twenty 96-well microplates. Ten days later, the culture supernatants were screened by ELISA for their ability to react with the purified MASP-2 factor.

ELISA analysis. Wells of Immunol® 2 microplates (Dynatech Laboratories, Chantilly, Va.) were coated by adding 50 μl of purified hMASP-2 or 50 ng/ml rat rMASP-2 (or rMASP-2A) overnight at room temperature. The low concentration of MASP-2 for coating allows for the selection of high affinity antibodies. After removing the coating solution by shaking the plate, 200 µl of BLOTTO (skimmed milk powder) in PBS was added to each well for one hour to block non-specific sites. After one hour, the wells were washed with PBST buffer (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants from each confluence were collected and mixed with 50 μl of BLOTTO and then added to individual microplate wells. After an hour of incubation, the wells were washed with PBST. Bound mouse 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 1:2000 in BLOTTO. A peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethylbenzidine (Sigma, Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) was added to the wells and allowed to stand for 30 minutes to develop color. The reaction was terminated by adding 50 μl of 2 M H ₂ SO ₄ to each well. The absorbance of the reaction mixture at 450 nm was determined using a BioTek ELISA Reader (BioTek® Instruments, Winooski, Vt.).

MASP-2 binding analysis.

Culture supernatants that test positive in the MASP-2 ELISA assay as described above can be tested in a binding assay to determine the binding affinity of MASP-2 inhibitory agents to MASP-2. A similar assay can be used to determine the ability of an inhibitory agent to bind to other antigens in the complement system.

Wells of a polystyrene microplate (96-well media binding plates, Corning Costar, Cambridge, MA) were coated with MASP-2 (20 ng/100 μl per well, Advanced Research Technology, San Diego, CA) in phosphate buffered saline (PBS), pH 7.4, overnight at 4°C. After removal 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 were used as controls. Aliquots of hybridoma supernatants or purified anti-MASP-2 MoAbs were added to the wells at varying concentrations in blocking solution. After a two hour incubation at room temperature, the wells were washed copiously with PBS. MASP-2-bound anti-MASP-2 MoAb was detected by adding peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) to the blocking solution, which was incubated for 1 hour at room temperature. The plate was rinsed again with PBS, after which 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added. The TMB reaction was stopped by adding 100 μl of 1 M phosphoric acid and the plate was read at 450 nm using a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

The culture supernatants from the positive wells were then tested for their ability to suppress complement activation in a functional assay such as the C4 cleavage assay as described in Example 2. Cells from the positive wells were then cloned by serial dilution. The MoAbs were again tested by ELISA for their ability to react with hMASP-2 as described above. Selected hybridomas were grown in spinner flasks and the depleted culture supernatant was collected for antibody purification by protein A affinity chromatography.

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Example 6

This example describes the preparation and production of humanized mouse aHTu-MASP-2 antibodies and antibody fragments.

A mouse anti-MASP-2 monoclonal antibody was obtained from male A/J mice as described in Example 5. The mouse antibody was then subjected to a humanization process as described below to reduce its immunogenicity by replacing mouse constant regions with human analogs to obtain chimeric IgG and An antibody Fab that can be used to suppress the side effects of MASP-2 dependent complement activation in humans according to the present invention.

1. Cloning of anti-MASP-2 variable region genes from mouse hybridoma cells. Total RNA was isolated from hybridoma cells secreting anti-MASP-2 MoAb (prepared as described in Example 7) using RNAzol according to 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 the first framework region of the mouse V _H or V _K genes as 5' primers. Anchored PCR was performed as described by Chen and Platsucas (Chen, PF, Scand. J. Immunol., 35:539-549, 1992). To clone the _VK gene, a double-stranded cDNA was prepared using primer Not1-MAK1 (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO: 38). Annealed adapters 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. Adapters at the 3' ends were removed by Not1 cleavage. The cleavage 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. DNA fragments of approximately 500 base pairs were cloned into the pUC19 vector. Several clones were selected for sequence analysis to confirm that the cloned sequence covered the desired murine immunoglobulin constant region. Oligonucleotides Not1-MAKl and MAK2 were obtained from the _VK region and located at 182 and 84 bp. respectively downstream from the first base pair of the C kappa gene. Clones were selected that contained the full length _VK and the leader peptide.

To clone the V _H gene, a double-stranded cDNA was prepared using primer Not1-MAG1 (1'CGCGGCCGCAGCTGCTCAGAGTGTAGA-5' SEQ ID NO: 42). The annealed AD1 and AD2 adapters were ligated to both the 5' and 3' ends of the double stranded cDNA. Adapters at the 3' ends were removed by Not1 cleavage. The cleavage product was then used as template in PCR with AD1 and MAG2 oligonucleotide (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. DNA fragments 500 to 600 bp in size. cloned into pUC19. The Not1-MAG1 and MAG2 oligonucleotides were obtained from the mouse Cy.7.1 region and located at 180 and 93 bp. respectively downstream from the first base pair in the mouse Cy.7.1 gene. Clones were selected that contained the full length VH and the leader peptide.

2. Construction of expression vectors for chimeric MASP-2 IgG and Fab. The cloned VH and VK genes described above were used as a template in a PCR reaction to add a Kozak consensus sequence to the 5' end and a splicing donor to the 3' end of the nucleotide sequence. After sequence analysis for the absence of PCR errors, the V _H and V _K genes were inserted into expression vector cassettes containing human C. y1 and C. kappa, respectively, to obtain pSV2neoVH-huCy1 and pSV2neoV-huCy. The heavy and light chain plasmid DNA vectors purified in a CsCl gradient were used to transfect COS cells by electroporation. After 48 hours of incubation, the culture supernatant was tested by ELISA to confirm the presence of about 200 ng/ml of chimeric IgG. Total RNA was isolated from the collected cells. First strand cDNA was synthesized from total RNA using oligo dT as a primer. This cDNA was used as a PCR template to generate DNA fragments for Fd and kappa. For the Fd gene, PCR was performed using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ ID NO: 44) as 5' primer and 3' primer isolated from CH1 (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO: 45) . The DNA sequence was confirmed to contain the complete VH and CH1 domain of human IgGl. After digestion with the appropriate enzymes, the Fd DNA fragments were inserted into the HindIII and BamHI restriction sites in the pSV2dhfr-TUS expression vector cassette to generate pSV2dhfrFd. Plasmid pSV2 is commercially available and consists of DNA segments obtained from various sources:

The pBR322 DNA (thin line) contains the pBR322 DNA origin (pBR ori) and the ampicillin lactamase resistance gene (Amp);

SV40 DNA, shown by broad strokes and labeling, contains the SV40 DNA origin of replication (SV40 ori), an early promoter (5' to the dhfr and neo genes), and a polyadenylation signal (3' to the dhfr and neo genes).

The polyadenylation signal derived from SV40 (pA) was also placed at the 3' end of the Fd gene.

- 59 043102

For the kappa gene, PCR was performed using 5'-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT3' (SEQ ID NO: 46) as the 5' primer and a 3' primer derived from CK (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 47). The DNA sequence was confirmed to contain full length VK and human CK regions. After digestion with the appropriate restriction enzymes, the kappa DNA fragments were inserted into the HindIII and BamHI restriction sites in the pSV2neo-TUS expression vector cassette to give pSV2neoK. Expression of both the Fd and kappa genes was initiated with an enhancer and promoter derived from HCMV. Since the Fd gene does not include a cysteine amino acid residue involved in the formation of a disulfide bond between the chains, this recombinant chimeric Fab contains non-covalently linked heavy and light chains. This chimeric Fab is referred to as cFab.

To obtain a recombinant Fab with a disulfide bond between the heavy and light chains, the above Fd gene can be extended by including a sequence encoding nine additional amino acids (EPKSCDKTH SEQ ID NO: 48) from the human IgG1 hinge region. The BstEII-BamHI DNA segment encoding the 30 amino acids at the 3' end of the Fd gene can be replaced with DNA segments encoding an extended Fd, resulting in pSV2dhfrFd/9aa.

3. Expression and purification of chimeric anti-MASP-2 IgG.

To obtain cell lines secreting chimeric anti-MASP-2 IgG, NSO cells were transfected with purified DNA plasmids pSV2neoVH-huC.y1 and pSV2neoV-huC kappa by electroporation. Transfected cells were selected in the presence of 0.7 mg/ml G418. Cells were grown in a 250 ml spinner flask with medium containing serum.

The culture supernatant obtained after centrifugation of 100 ml of 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 50 mM citrate buffer, pH 3.0. An equal volume of 1 M Hepes, pH 8.0 was added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts were removed by buffer exchange with PBS by ultrafiltration through a Millipore membrane (cut-off MW: 3000). The protein concentration of the purified antibody was determined by the BCA method (Pierce).

4. Expression and purification of the chimeric anti-MASP-2 Fab.

To obtain cell lines secreting chimeric anti-MASP-2 Fab, CHO cells were transfected with purified DNA plasmids pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa by electroporation. Transfected cells were selected in the presence of G418 and methotrexate. Selected cell lines were expanded at elevated concentrations of methotrexate. The cells were single cells subcloned by serial dilution. Then, highly productive cell lines obtained from subcloned single cells were grown in 100 ml of culture in a serum-free medium with constant stirring.

Chimeric anti-MASP-2 Fab was purified by affinity chromatography using mouse anti-idiotypic MoAb to MASP-2 MoAb. An anti-idiotypic MASP-2 MoAb can be generated by immunizing mice with mouse anti-MASP-2 MoAb conjugated to keyhole hemocyanin (KLH) and then screened for specific MoAb binding that can compete with human MASP-2. For purification, 100 ml of supernatant from culture of CHO cells producing cFab or cFab/9aa was loaded onto an affinity column with bound anti-idiotypic MASP-2 MoAb. The column was then thoroughly washed with PBS until the bound Fab was eluted with 50 mM diethylamine, pH 11.5. Residual salts were removed by buffer exchange as described above. The protein concentration of the purified Fab was determined by the BCA method (Pierce).

The ability of chimeric MASP-2 IgG, cFab and cFAb/9aa to suppress the MASP-2 dependent complement activation pathway can be determined by the suppression assay described in Example 2.

Example 7

This example describes an in vitro C4 cleavage assay used as a functional screen to identify MASP-2 inhibitory agents capable of inhibiting MASP-2 dependent activation using L-ficolin/P35, H-ficolin, M-ficolin, or mannan.

C4 cleavage analysis. C4 cleavage assay is described in Petersen et al., J. Immunol. Methods, 257:107 (2001), which measured lectin pathway activation initiated by lipoteichoic acid (LTA) from S. aureus, which binds to L-ficolin.

Reagents: Formalin-fixed S. aureous (DSM20233) was prepared as follows: bacteria were grown overnight at 37°C in trypton soy agar medium supplemented with blood, washed three times with PBS, then fixed for 1 h at room temperature in PBS/0.5% formalin and washed three times with PBS before resuspension in immobilization buffer (15 mM Na ₂ CO 3 , 35 mM NaHCO 3 , pH 9.6).

Analysis. The wells of a Nunc MaxiSorb microplate (Nalgene Nunc International, Rochester, NY) were coated with 100 μl formalin-fixed S. aureus DSM20233 (00550=0.5) in immobilization buffer with 1 μg L-ficolin. After overnight incubation, wells are blocked with 0.1% human serum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then washing

- 60 043102 whether TBS containing 0.05% Tween 20 and 5 mm CaCl ₂ (wash buffer). Human serum samples were dissolved in 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevented activation of endogenous C4 and led to dissociation of the complex C1 (consisting of C1q, C1r and C1s). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides, were added to serum samples at various concentrations. Diluted samples were applied to the plate and incubated overnight at 4°C. After 24 h, the plates were thoroughly washed with wash buffer, then 0.1 μg of purified human C4 (prepared as described in Dodds, AW, Methods Enzymol., 223:46, 1993) in 100 μl of 4 mM barbital, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4. After 1.5 h incubation at 37°C, the plates were washed again and C4b deposition was determined using alkaline phosphatase-conjugated chicken anti-human C4c (Immunsystem, Uppsala, Sweden) and measured colorimetrically with pnitrophenyl phosphate substrate.

Analysis of C4 on mannan. The above assay was adapted to measure lectin pathway activation by MBL by coating the plate with LPS and mannan before adding serum mixed with various MASP-2 inhibitory agents.

C4 analysis for H-ficolin (Hakata Ag). The above assay was adapted to measure lectin pathway activation by H-ficolin by coating the plate with LPS and H-ficolin before adding serum mixed with various MASP-2 inhibitory agents.

Example 8

The following analysis demonstrates the presence of classical pathway activation in wild-type and MASP-2 -/- mice.

Methods. Immune complexes were prepared in situ by coating microplates (Maxisorb, Nunc, cat. No 442404, Fisher Scientific) with 0.1% human serum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 h at room temperature, followed by overnight incubation at 4° C. with sheep anti-whole antiserum (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 with TBS/Tween/Ca ²⁺ . Serum samples were obtained from wild-type mice and MASP-2 -/- mice and added to coated tablets. Prepared control samples in which C1q was extracted from serum samples of wild-type mice and MASP-2 -/- mice. C1q-depleted mouse serum was prepared using Dynabeads® protein-A coupled microspheres (Dynal Biotech, Oslo, Norway) coated with C1q rabbit anti-human IgG (Dako, Glostrup, Denmark) according to the manufacturer's instructions. The plates were incubated for 90 min at 37°C. Bound C3b was detected with a polyclonal anti-human C3c antibody (Dako A 062) diluted 1:1000 in TBS/Tween/Ca ⁺⁺ . The secondary antibody was goat anti-rabbit IgG.

Results. In FIG. 9 shows the relative levels of C3b deposition on IgG coated plates in wild-type serum, MASP-2-/- serum, C1q-depleted wild-type serum, and C1q-depleted MASP-2-/- serum. These results indicate that the MASP-2-/classic pathway remains intact in mice.

Example 9

The following assay was used to test the ability of a MASP-2 inhibitory agent to block the classical activation pathway by examining the effect of the MASP-2 inhibitory agent under conditions of classical pathway initiation by immune complexes.

Methods.

To assess the effect of an inhibitory MASP-2 agent under complement activation conditions, in which the classical pathway is initiated by immune complexes, three serum samples, 50 µl each, containing 90% NHS, were incubated at 37°C in the presence of 10 µg/ml immune complex ( IC) or PBS, and in parallel incubated three more samples (+/-IC) containing 200 nm antiproperdin monoclonal antibodies at 37°C. After a two hour incubation at 37°C, 13 mM EDTA was added to all samples to stop further complement activation, after which all samples were immediately cooled to 5°C. The samples were then stored at -70° C. until used in the assay for complement activation products (C3a and sC5b-9) using an ELISA kit (Quidel cat. no. A015 and A009) according to the manufacturer's instructions.

Example 10

This example describes the identification of high affinity Fab2 fragments of anti-MASP-2 antibodies that block MASP-2 activity.

Background and Rationale: MASP-2 is a protein complex with many distinct functional domains including binding site(s) for MBL and ficolins, serine protease catalytic site, C2 proteolytic substrate binding site, C4 proteolytic substrate binding site, MASP cleavage site -2 for self-activation of the MASP-2 zymogen and two Ca ^++- binding sites. Fab2 fragments of antibodies that are high affinity for MASP-2 have been identified, and these identified Fab2 fragments have been tested in a functional assay to determine their ability to block functionality.

- 61 043102 nuyu activity of MASP-2.

To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind or interfere with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Therefore, many or all high affinity anti-MASP-2 binding Fab2s do not have the ability to suppress the functional activity of MASP-2 unless they bind to structural epitopes on MASP-2 that are directly involved in the functional activity of MASP-2.

A functional assay measuring inhibition of lectin pathway C3 convertase production was used to evaluate the blocking activity of anti-MASP-2 Fab2. It is known that the primary physiological role of MASP-2 in complement activation via the lectin pathway is to form the next functional component of lectin-dependent complement activation, known as lectin pathway C3 convertase. The lectin pathway C3 convertase is a critical enzyme complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, functional activity of MASP-2 is required for the formation of two protein components (C4b, C2a) that contain the lectin pathway C3 convertase. In addition, all of the individual functional activities of MASP-2 listed above are required for the formation of the lectin pathway C3-convertase with the participation of MASP-2. For this reason, the preferred assay method for assessing the blocking activity of anti-MASP-2 Fab2 is considered to be a functional assay that allows measurement of the suppression of the formation of C3 convertase of the lectin pathway.

Formation of high affinity Fab2. To generate high affinity Fab2s for the rat MASP-2 protein (SEQ ID NO: 55), a phage display library of human antibody light and heavy chain sequences and automated antibody selection technology was used to identify Fab2s that react with selected ligands of interest. A known amount of rat MASP-2 protein (~1 mg, >85% purity) was used for antibody screening. Three cycles of amplification were used to select antibodies with the best affinity. Approximately 250 different variants expressing antibody fragments were selected for screening by ELISA. High affinity variants were subsequently sequenced to determine the uniqueness of the various antibodies.

Fifty unique anti-MASP-2 antibodies were purified and 250 μg of each purified Fab2 antibody was used for MASP-2 binding affinity characterization and complement pathway functional testing, as described in more detail below.

Assays used to evaluate the inhibitory (blocking) activity of anti-MASP-2 Fab2.

1. Assay to measure the inhibition of the formation of C3-convertase of the lectin pathway.

Prerequisites. The lectin pathway C3 convertase is an enzymatic complex (C4bC2a) that proteolytically cleaves C3 into two potential pro-inflammatory fragments, anaphylatoxin C3a and opsonin C3b. The formation of C3 convertase is a key step in the lectin pathway in terms of mediating inflammation. MASP-2 is not a structural component of the C3 convertase (C4bC2a) of the lectin pathway, so anti-MASP-2 antibodies (or Fab2) will not directly suppress the activity of the pre-existing C3 convertase. However, the production of two protein components (C4b, C2a) that contain the C3 convertase of the lectin pathway requires the activity of the serine protease MASP-2. Therefore, anti-MASP-2 Fab2s that suppress the functional activity of MASP-2 (ie, block anti-MASP-2 Fab2) will suppress de novo formation of the lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioether group in its structure. When C3 is cleaved with C3 convertase in this assay, the thioether group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of the plastic wells via ester or amide bonds, which facilitates detection of C3b by ELISA.

Yeast mannan is known to be a lectin pathway activator. In the following method for measuring C3 convertase production, mannan-coated plastic wells were incubated for 30 min at 37°C with diluted rat serum to activate the lectin pathway. The wells were then washed and analyzed for the presence of C3b immobilized in the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of the lectin pathway C3 convertase. Ahtu-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b formation.

Methods.

Costar Medium Binding 96-well plates were incubated overnight at 5° C. with mannan diluted in 50 mM carbonate buffer (pH 9.5) at 1.0 μg/50 μl/well. After overnight incubation, each well was washed three times with 200 μl PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated for 1 hour at room temperature with gentle agitation. Each well was washed three times with 200 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations with GVB buffer containing Ca++ and Mg ⁺⁺ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin , pH 7.4) at 5°C. In the above image

0.5% rat serum was added at 5°C and 100 µl was added to each well. The plates were capped and incubated for 30 min in a water bath at 37°C to activate complement. The reaction was stopped by transferring the plates from the 37° C. water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 μl PBS-Tween 20 (0.05% Tween 20 in PBS), then washed twice with 200 μl PBS. The primary antibody (rabbit anti-human C3c, DAKO A0062) at a 1:10,000 dilution at 100 μl/well was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 hour at room temperature with gentle agitation. Each well was washed five times with 200 μl PBS. The secondary antibody (perxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) at a 1:10,000 dilution of 100 µl/well was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 h at room temperature for shaker with gentle stirring. Each well was washed five times with 200 μl PBS. TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added at 100 µl/well and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 1.0 M H3PO4 at 100 μl/well and the OD ₄₅₀ was measured.

2. Assay to measure suppression of MASP-2 dependent C4 cleavage.

Prerequisites. MASP-2 serine protease activity is highly specific and only two protein substrates have been identified for MASP-2, C2 and C4. When C4 is cleaved, C4a and C4b are formed. Ahtu-MASP-2 Fab2 can bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (eg, MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thus inhibit functional activity MASP-2 leading to C4 cleavage.

Yeast mannan is known to be a lectin pathway activator. In the method described below for measuring MASP-2 C4 cleavage activity, mannan-coated plastic wells were incubated for 30 minutes at 37°C with diluted rat serum to activate the lectin pathway. Since the primary antibodies used in this ELISA only recognize C4, diluted rat serum was also supplemented with human C4 (1.0 μg/ml). The wells were then washed and analyzed for the presence of human C4b immobilized on the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2 dependent C4 cleavage activity. Ahtu-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.

Methods. Costar Medium Binding 96-well plates were incubated overnight at 5° C. with mannan diluted in 50 mM carbonate buffer (pH 9.5) at 1.0 μg/50 μl/well. Each well was washed three times with 200 μl PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated for 1 hour at room temperature with gentle agitation. Each well was washed three times with 200 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations with GVB buffer containing Ca++ and Mg ⁺⁺ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin , pH 7.4) at 5°C. These samples were also injected with 1.0 μg/ml human C4 (Quidel). In the above samples, 0.5% rat serum was added at 5° C. and 100 μl was added to each well. The plates were capped and incubated for 30 min in a water bath at 37°C to activate complement. The reaction was stopped by transferring the plates from the 37° C. water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 μl PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed twice with 200 μl PBS. Biotin-conjugated chicken anti-human C4 (Immunsystem AB, Uppsala, Sweden) at a 1:700 dilution at 100 µl/well was added to PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated for 1 h at room temperature with gentle stirring. Each well was washed five times with 200 μl PBS. 0.1 μg/ml peroxidase-conjugated streptavidin (Pierce Chemical #21126) at 100 μl/well was added to PBS containing 2.0 mg/ml BSA and incubated for 1 hour at room temperature on a shaker with gentle agitation. Each well was washed five times with 200 μl PBS. TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added at 100 µl/well and incubated at room temperature for 16 minutes. Peroxidase reaction was stopped by adding 1.0 M H ₃ PO ₄ in the amount of 100 μl/well and measured OD ₄₅₀ .

3. Anti-rat MASP-2 Fab2 binding assay to native rat MASP-2.

BACKGROUND: MASP-2 is normally present in plasma in the form of dimeric MASP-2 complexes, which also include specific lectin molecules (mannose-binding protein (MBL) and ficolins). As a consequence, if there is interest in studying the binding of anti-MASP-2 Fab2 to a physiologically relevant form of MASP-2, it is important to develop a binding assay that uses the interaction of Fab2 not with purified recombinant MASP-2, but with native plasma MASP-2. In this binding assay, native MASP-2-MBL complex from 10% rat serum is first immobilized in mannan-coated wells. The binding affinity of various anti-MASP-2 Fab2s for immobilized native MASP-2 is then studied by standard ELISA.

- 63 043102

Methods. Costar High Binding 96-well plates were incubated overnight at 5° C. with mannan diluted in 50 mM carbonate buffer (pH 9.5) at 1/50 μl/well. Each well was washed three times with 200 μl PBS. The wells were blocked with 0.5% skimmed milk powder in PBST (PBS with 0.05% Tween 20) at 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. Each well was washed three times with 200 μl TBS/Tween/Ca ⁺⁺ wash buffer (tris buffered saline, 0.05% Tween 20 containing 5.0 mM CaCl2, pH 7.4). 10% rat serum was prepared on ice in high salt immobilization buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCl2, 0.05% Triton X100, 0.1% (w/v) bovine serum albumin, pH 7 ,4). 100 μl/well was added and incubated overnight at 5°C. The wells were washed three times with 200 μl TBS/Tween/Ca ⁺⁺ wash buffer. The wells were then washed twice with 200 μl PBS. 100 µl/well of selected concentration of anti-MASP-2 Fab2 diluted in GVB buffer containing Ca ⁺⁺ and Mg ⁺⁺ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCb, 2.0 mM CaCL 0, 1% gelatin, pH 7.4) was added and incubated for 1 hour at room temperature with gentle agitation. Each well was washed five times with 200 μl PBS. HRP-conjugated goat anti-Fab2 (Biogenesis cat. no. 0500-0099) diluted 1:5000 in 2.0 mg/mL BSA in PBS was added at 100 μg/well and incubated for 1 h at room temperature with gentle stirring. Each well was washed five times with 200 μl PBS. TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added at 100 µl/well and incubated at room temperature for 70 minutes. The peroxidase reaction was stopped by adding 1.0 M H3PO ₄ at 100 μl/well and the OD ₄₅₀ was measured.

Results.

Approximately 250 different Fab2s that reacted with high affinity with the rat MASP-2 protein were selected for screening by ELISA. These high affinity Fab2s were sequenced to determine the uniqueness of the various antibodies and 50 unique anti-MASP-2 antibodies were purified for further analysis. 200 μg of each purified Fab2 antibody was used for MASP-2 binding affinity characterization and complement pathway functional testing. The results of this analysis are presented below in table. 6.

Table 6

Ahtu-MASP-2 Fab2 blocking complement activation via the lectin pathway

No. of antibody Fab2 C3-convertase (1C ₅₀ (nM) _Kd Cleavage C4 1C ₅₀ (nM) 88 0.32 4.1 ND 41 0.35 0.30 0.81 eleven 0.46 0.86 <2 nM 86 0.53 1.4 ND 81 0.54 2.0 ND 66 0.92 4.5 ND 57 0.95 3, 6 <2 nM 40 1.1 7.2 0.86 58 1.3 2, 6 ND 60 16 3, 1 ND 52 16 5, 8 <2 nM 63 2.0 6, 6 ND 49 2.8 8.5 <2 nM 89 thirty 2.5 ND 71 thirty 10.5 ND 87 6.0 2.5 ND 67 100 7.7 ND

As shown above in Table. 6, of 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2s that could potentially inhibit the production of C3 convertα with an IC ₅₀ equal to or less than 10 nmol Fab2 (34% positive success rate) . Eight of the seventeen identified Fab2 have an IC ₅₀ value in the subnanomolar region. In addition, all seventeen Fab2, blocking MASP-2, shown in table. 6 essentially demonstrate complete inhibition of C3 convertase production in the lectin pathway C3 convertase assay. In FIG. 8A is a graphical representation of the results of the C3 convertase production assay for the Fab2 antibody.

- 64 043102 No. 11, which is a typical representative of the tested Fab2 antibodies, the results of which are shown in table. 6. This fact is important because it suggests the theoretical possibility that blocking Fab2 may only partially suppress MASP-2 function even when every MASP-2 molecule binds to Fab2.

Although mannan is a known activator of the lectin pathway, there is a theoretical possibility that the presence of anti-mannan antibodies in rat serum could also activate the classical pathway and generate C3b via the classical pathway C3 convertase. However, each of the seventeen blocking anti-MASP-2 Fabs listed in this example could potentially inhibit C3b production (>95%), thus demonstrating the specificity of this assay for the lectin pathway C3 convertase.

Binding analysis was also performed on all seventeen blocking Fab2s to calculate the apparent Kd for each of them. The results of the analysis of binding of anti-rat MASP-2 Fab2 to native rat MASP-2 for six blocking Fab2 are also presented in table. 6. In FIG. 8B is a graphical representation of the results of the Fab2 antibody #11 binding assay. 6. Basically, the apparent Kd obtained for the binding of each of the six Fab2 to native MASP-2, fully consistent with the IC ₅₀ for Fab2 in the functional analysis of C3 convertase. There is evidence that MASP-2 undergoes a conformational change from an inactive to an active form upon activation of its protease activity (Feinberg et al., EMBO J., 22:2348-59 (2003); Gal et al., J. Biol. Chem ., 280:33435-44 (2005)). In normal rat plasma used in the C3 convertase production assay, MASP-2 is present primarily 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 mannan; therefore, MASP-2 is rather an active conformation (Petersen et al., J. Immunol Methods, 257:107-16, 2001). Therefore, a clear relationship between IC ₅₀ and K _d for each of the seventeen blocking Fab2s tested in these two functional assays should not be expected, since Fab2 will bind to different conformational forms of MASP-2 in each assay. However, with the exception of Fab2 #88, there appears to be a fairly close match between IC ₅₀ and apparent K _d for each of the remaining sixteen Fab2s tested in two tests (see Table 6).

Some blocking Fab2 were evaluated for the ability to suppress MASP-2-dependent C4 cleavage. In FIG. 8C graphically shows the results of the C4 cleavage assay showing suppression for Fab2 #41, with IC ₅₀ =0.81 nM (see Table 6). As shown in FIG. 9, all of the Fab2s tested suppressed C4 cleavage with similar IC ₅₀ values to those obtained in the C3 convertase assay (see Table 6).

Although mannan is a known activator of the lectin pathway, there is a theoretical possibility that the presence of anti-mannan antibodies in rat serum could also activate the classical pathway and thus generate C4b through C1s-mediated C4 cleavage. However, several anti-MASP-2 Fab2s have been identified that effectively suppress C4b production (>95%), demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioether group in its structure. In this assay, when C4 is cleaved by MASP-2, the thioether group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of the plastic wells through ester or amide bonds, which facilitates the detection of C4b by ELISA.

These studies clearly demonstrate the creation of high affinity Fab2s for the rat MASP-2 protein, which functionally block both C4 and C3 convertase activity, thereby preventing activation of the lectin pathway.

Example 11.

This example describes epitope mapping of several blocking anti-rat MASP-2 antibodies Fab2 obtained as described in example 10.

Methods.

As shown in FIG. 10, the following proteins, all with N-terminal 6X His tags, were expressed in CHO cells using the pED4 vector:

rat MASP-2A, a full-length MASP-2 protein inactivated by alanine substitution at the active site of serine (S613A);

rat MASP-2K, a full-length MASP-2 protein modified to reduce autoactivation (R424K);

CUBI-II, an N-terminal fragment of rat MASP-2 that contains only CUBI, EGF-like and CUBII domains; And

CUBI/EGF-like, N-terminal fragments of rat MASP-2 that contain only CUBI and EGF-like domains.

These proteins were purified from culture supernatants by nickel affinity chromatography as

- 65 043102 described previously (Chen et al., J. Biol. Chem., 276:25894-02 (2001)).

A C-terminal polypeptide (CCPII-SP) containing CCPII and a rat MASP-2 serine protease domain was expressed in E. coli as a thioredoxin fusion protein using pTrxFus (Invitrogen). Protein was purified from cell lysates using Thiobond affinity resin. The thioredoxin fusion protein was expressed in empty pTrxFus as a negative control.

All recombinant proteins were dialyzed in TBS buffer and their concentrations were determined by measuring the OD at 280 nm.

Dot blot analysis.

Serial dilution of the five recombinant MASP-2 polypeptides described above and shown in FIG. 10 (and a thioredoxin polypeptide as a negative control for the CCPII-serine protease polypeptide) were applied to a nitrocellulose membrane. The amount of protein applied was in the range from 100 ng to 6.4 pg, in five-fold dilutions. In subsequent experiments, the amount of applied protein was in the range from 50 ng to 16 pg, also in five-fold dilutions. The membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer), then incubated with 1.0 μg/ml anti-MASP-2 Fab2 in blocking buffer (containing 5.0 mm Ca ²⁺ ). Bound Fab2s were detected using an HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL detection kit (Amersham). One membrane was incubated with a polyclonal rabbit anti-human MASP-2 Ab (described in Stover et al., J. Immunol., 163:684859 (1999)) as a positive control. In this case, bound Ab was detected with HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2000).

MASP-2 binding analysis.

ELISA plates were coated with recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) at 1.0 μg per well overnight at 4°C. The wells were blocked with 1% BSA in TBS, then serial dilutions of anti-MASP-2 Fab2 in TBS containing 5.0 mM Ca ²⁺ were added. The plates were incubated for 1 h at room temperature. After a triple wash with TBS/Tween/Ca ²⁺ , HRP-conjugated anti-human Fab (AbD/Serotec) diluted 1/10,000 in TBS/Ca ²⁺ was added and the plates were again incubated for 1 hour at room temperature. Bound antibody was detected using the TMB kit, a peroxidase substrate (Biorad).

Results.

The results of the dot-blot analysis, showing the reactivity of Fab2 in relation to various MASP-2 polypeptides, are presented below in table. 7. Numerical values given in Table. 7 denote the amount of applied protein required to obtain a signal with an intensity of about 1/2 of the maximum value. As can be seen, the positive control Ab (polyclonal anti-human MASP-2 serum obtained from rabbits) recognized all polypeptides (the only exception being the thioredoxin chimeric protein).

Table 7

Reactivity with various recombinant rat masp-2 polypeptides in dot blot analysis

No. of antibody Fab2 MASP-2A CUBI II CUBI/EGF-like CCPII- SP Thyroidoxin 40 0.16 ng NR NR 0.8 ng NR 41 0.16 NG NR NR 0.8 ng NR eleven 0.16 ng NR NR 0.8 ng NR

- 66 043102

49 0.16 ng NR 52 0.16 ng NR 57 0.032 ng NR 58 0.4 ng NR 60 0.4 ng 0.4 ng 63 0.4 ng NR 66 0.4 ng NR 67 0.4 ng NR 71 0.4 ng NR 81 0.4 ng NR 86 0.4 ng NR 87 0.4 ng NR Positive control <0.032 ng 0.16 ng

NR >2 0 ng NR NR 0.8 ng NR NR NR NR NR 2.0 ng NR NR NR NR NR 2.0 ng NR NR 2.0 ng NR NR 2.0 ng NR NR 2.0 ng NR NR 2.0 ng NR NR 10 ng NR NR 2.0 ng NR 0.16 ng <0.032 ng NR

NR=Hem reactions.

The positive control antibody is polyclonal anti-human MASP-2 serum obtained from rabbits.

All Fab2s reacted with MASP-2A as well as MASP-2K (data not shown). Most Fab2s recognized the CCPII-SP polypeptide but not the N-terminal fragments. Exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and a CUBI-II fragment, but does not recognize a CUBI/EGF-like polypeptide or a CCPII-SP polypeptide, suggesting its binding to an epitope on CUBII, or an overlap of CUBII and an EGF-like domain. Fab2 #57 recognizes MASP-2A but does not recognize any of the MASP-2 fragments tested, indicating that this Fab2 antibody recognizes an epitope on CCPI. Fab2 #40 and 49 only bind to complete MASP-2A. In the ELISA binding assay shown in FIG. 11, Fab2 #60 also bound to the CUBI-II polypeptide, although with a slightly lower apparent affinity.

These results demonstrate the unique ability of Fab2 to block multiple regions of the MASP-2 protein.

Example 12.

This example describes the identification by phage display of fully human scFv antibodies that bind to MASP-2 and suppress lectin-mediated complement activation without affecting the classical (C1 q-dependent) immune system component pathway.

Short review.

Fully human, high affinity MASP-2 antibodies were identified by screening the phage display library. Fragments of the variable regions of the light and heavy chains of antibodies were isolated both in scFv format and in full-length IgG format. Human antibodies to MASP-2 are useful in suppressing cellular damage associated with lectin-mediated activation of the complement pathway without affecting the classical (C1c-dependent) component of the immune system. In some embodiments, the provided MASP-2 inhibitory antibodies have the following characteristics:

(a) high affinity for human MASP-2 (eg, KD=10 nM or less); and (b) inhibit MASP-2 dependent complement activity in 90% human serum with an IC ₅₀ of 30 nM or less.

Methods.

Expression of full length catalytically inactive MASP-2.

The full-length human MASP-2 cDNA sequence (SEQ ID NO: 4) 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 expression in eukaryotes under 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, 185:531-66 (1991)).

To obtain a catalytically inactive human MASP-2A protein, site-directed mutagenesis was performed as described in US 2007/0172483, incorporated herein by reference. PCR products were purified after agarose gel electrophoresis and banding, and single adenosine overlaps were obtained using a standard extension procedure. Adenosine-supplemented MASP-2A was then cloned into the 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 above expression construct MASP-2 and MASP-2A were transfected into cells

- 67 043102

DXB1 using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was obtained in a serum-free nutrient medium to avoid contamination of preparations with other serum proteins. A day later, confluent cells were collected from nutrient media (in total, the process was carried out four times). The average level of recombinant MASP-2A was about 1.5 mg/l culture medium. MASP-2A (the Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns.

ELISA method for MASP-2A on candidate ScFv clones identified by panning/scFv transformation and filter screening.

A phage display library of human immunoglobulin light and heavy chain variable sequences was subjected to antigenic panning followed by automated screening and selection to detect high affinity scFv antibodies to the human MASP-2 protein. The scFv phage library was panned three times against the HIS tag or biotin-labeled MASP-2A. The third round of panning was eluted first with MBL and then with TEA (alkaline). To monitor the specific enrichment of phages displaying scFv fragments relative to the target MASP-2A, polyclonal phage ELISA was performed against immobilized MASP-2A. The scFv genes from the 3 panning cycle were cloned into the pHOG expression vector and screened through a fine filter for specific clones targeting MASP-2A.

Bacterial colonies containing plasmids encoding scFv fragments from cycle 3 panning were collected, placed on nitrocellulose membranes and grown overnight on non-inducing medium to obtain master plates. A total of 18,000 colonies were selected and analyzed from the 3 panning cycle, half from competitive elution and half from subsequent TEA elution. Panning the anti-MASP-2A phagemid scFv library followed by scFv conversion and filter screening yielded 137 positive clones. 108/137 clones were positive in the ELISA for MASP-2 binding (data not shown), of which 45 clones were further tested for their ability to block MASP-2 activity in normal human serum.

Assay to measure inhibition of lectin pathway C3 convertase production.

A functional assay measuring inhibition of lectin pathway C3 convertase production was used to evaluate the blocking activity of candidate MASP-2 scFv clones. To obtain two protein components (C4b, C2a), which contain the C3-convertase of the lectin pathway, the activity of the serine protease MASP-2 is required. Therefore, MASP-2 scFvs that inhibit MASP-2 functional activity (ie, block MASP-2 scFvs) will suppress de novo formation of the lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioether group in its structure. When C3 is cleaved with C3 convertase in this assay, the thioether group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of the plastic wells via ester or amide bonds, which facilitates detection of C3b by ELISA.

Yeast mannan is known to be a lectin pathway activator. In the following method for measuring C3 convertase production, mannan-coated plastic wells were incubated with diluted human serum to activate the lectin pathway. The wells were then washed and analyzed for the presence of C3b immobilized in the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of the lectin pathway C3 convertase. MASP-2 scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase production and subsequent C3b production.

Methods.

candidate clones identified as described above were expressed, purified and diluted to the same stock concentration, which was diluted again with GVB buffer containing Ca++ and Mg ^- (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) to ensure that all clones have the same amount of buffer. scFv clones were tested in triplicate at a concentration of 2 μg/ml. The positive control was OMS100 Fab2, which was tested at a concentration of 0.4 μg/ml. C3c formation was monitored in the presence and absence of scFv/IgG clones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mM carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO ₃ +l.5 mM NaN ₃ ), pH 9.5, and covered the ELISA plates with it, leaving it on overnight at 4°C. The next day, the mannan-coated plates were washed 3 times with 200 μl PBS. Then, 100 µl of 1% HSA blocking solution was added to the wells and incubated for 1 h at room temperature. The plates were washed three times with 200 μl PBS and kept on ice with 200 μl PBS until samples were added.

Normal human serum was diluted to 0.5% concentration in CaMgGVB buffer and scFv clones or OMS100 Fab2 positive control were added in triplicate at 0.01, 1 µg/mL (OMS100 control only) and 10 µg/mL and pre-incubated for 45 min on ice before adding to the blocked ELISA plate. The reaction was initiated by incubation

- 68 043102 ee for 1 hour at 37°C, which was stopped by transferring the tablets to an ice bath. C3b deposition was detected with a rabbit antibody to α-mouse C3c followed by an HRP-conjugated goat anti-os-rabbit antibody. The negative control was antibody-free buffer (no antibodies=maximum C3b deposition), and the positive control was EDTA buffer (no C3b deposition). Background was determined using the same assay, except that the wells did not contain mannan. The background signal relative to the plates without mannan was subtracted from the signals in the mannan-containing wells. The cut-off criterion was set to half the activity of the irrelevant scFv clone (VZV) and pure buffer.

Results. Based on the cut-off criteria, a total of 13 clones were found to block MASP-2 activity. All 13 clones providing >50% pathway suppression were selected and sequenced to give 10 unique clones. All ten clones were found to have a light chain of the same subclass, λ3, but a heavy chain of three different subclasses: VH2, VH3 and VH6. In functional analysis, five out of ten candidate scFv clones had IC ₅ o values in nM below the target criterion, 25 nM using 0.5% human serum.

To detect antibodies with improved performance, the three maternal scFv clones identified as described above were subjected to light chain shuffling. This process involved the creation of a combinatorial library consisting of the Vh of each maternal clone paired with a library of native human lambda light chains (V _L ) obtained from six healthy donors. This library was then screened for scFv clones with improved binding affinity and/or functionality.

Table 8

Comparison of functional efficiency according to 1C ₅ o (nM) of the main daughter clones and their corresponding maternal clones (all in scFv format)

scFv-clone 1% human serum, C3 assay (1C ₅₀ nM) 90% human serum, C3 assay (IC50 nM) 90% human serum, C4 assay (IC50 nM) 17D20mc 38 Nd Nd 17D20m_d3521Nl 1 26 >1000 140 17N16mc 68 Nd Nd 17N16m_dl7N9 48 15 230

Below are the heavy chain variable region (VH) sequences for the parent clones and daughter clones shown in Table 1 above. 8.

CDRs (31-35 (Hl), 50-65 (H2) and 95-107 (H3)) by Kabat are shown in bold; and CDRs (26-32 (Hl), 52-56 (H2) and 95-101 (H3)) by Chothia are underlined.

Heavy chain variable region (VH) 17D2O_35VH-21N11VL (SEQ ID NO: 67, encoded by SEQ ID NO: 66).

QVT LKE S G PVLVKP T E T L T L T C TVSGFSLSRGKMGVSWIRQP PGKALEWLAHIFSSDEK

SYRTSLKSRLTISKDTSKNQWLTMTNMDPVDTATYYCARIRRGGIDYWGQGTLVTVSS

Heavy chain variable region (VH) dl7N9 (SEQ ID NO: 68).

QVQLQOSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWLGRTYYRSKW

YNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDPFGVPFDIWGOGTMVTVSS

Below are the light chain variable region (VL) sequences for the parent clones and daughter clones shown above in Table 1. 8.

CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) by Kabat are in bold; and CDRs (24-34 (L1); 50-56 (L2) and 89-97 ( L3) Chothia underlined These sections are the same regardless of the Kabat or Chothia numbering system.

Light chain variable region (VL) 17D20m_d3521Nl 1 1 (SEQ ID NO: 69, encoded by SEQ ID NO: 70).

QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQS PVLVMYQDKQRPSGIPE

RFSGSNSGNTATLTISGTOAMDEADYYCQAWDSSTAVFGGGTKLTVL

Light chain variable region (VL) 17N16m_dl7N9 (SEQ ID NO: 71).

SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYOQRPGQAPVLVIYDDSDRPSGIPD

RFSASNSGNTATLTITRGEAGDEADYYCOyWDIATDHWFGGGTKLTVLAAAGSEQKLISE

Ahth-MASP-2 antibodies OMS100 and MoAb_d3521NllVL (containing the variable region of the

-69043102 light chain shown in SEQ ID NO: 67 and light chain variable region shown in SEQ ID NO: 69, also referred to as OMS646 and mAb6), both showing high affinity binding of human MASP-2 and having the ability to block functional activity complement, analyzed for epitope binding by dot blot analysis. The results show that the antibodies OMS646 and OMS100 are highly specific for MASP-2 and do not bind to MASP-1/3. None of the antibodies bound to MAp19 or MASP-2 fragments contained the MASP-2 CCP1 domain, suggesting that the binding sites included CCP1.

The anti-MASP-2 antibody OMS646 was determined to actively bind to recombinant MASP-2 (Kd 60 to 250 μM) with a selectivity 5000 times greater than that for binding C1s, C1r or MASP-1 (see Table 9). below).

Table 9

Binding affinity and specificity of the interaction of anti-MASP-2 antibody OMS646 with MASP-2, according to the results of solid-phase ELISA

Antigen K _D (pM) MASP-1 >500,000 MASP-2 62+23* MASP-3 >500,000 Purified human C1r >500,000 Purified Human Cis -500,000

* Mean±SD; n=12.

OMS646 specifically blocks the components of lectin-dependent activation.

Methods.

The effect of OMS646 on membrane attack complex (MAC) deposition was analyzed using specific conditions for the lectin pathway, the classical pathway, and the alternative pathway. For this purpose, the Wieslab Comp300 Complement Screening Kit (Wieslab, Lund, Sweden) was used according to the manufacturer's instructions.

Results.

In FIG. 12A is a graphical representation of the level of MAC deposition in the presence or absence of antiMASP-2 antibody (OMS646) under lectin pathway specific conditions. In FIG. 12B is a graphical representation of the level of MAC deposition in the presence or absence of an anti-MASP-2 antibody (OMS646) under classical pathway-specific conditions. In FIG. 12C is a graphical representation of the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway specific conditions.

As shown in FIG. 12A, OMS646 blocks lectin-mediated activation of MAC deposition with an IC50 value of about 1 nM. However, OMS646 did not affect MAC deposition resulting from activation mediated by the classical pathway (FIG. 12B) or the alternative pathway (FIG. 12C).

Pharmacokinetics and pharmacodynamics of OMS646 after intravenous (IV) or subcutaneous (SC) administration in mice

The pharmacokinetics (PK) and pharmacodynamics (PD) of OMS646 were evaluated in a 28 day single dose PK/PD study in mice. The study tested dose levels of 5 and 15 mg/kg OMS646 administered subcutaneously (SC), as well as a dose level of 5 mg/kg OMS646 administered intravenously (IV).

With respect to the PK profile of OMS646, FIG. 13 is a graphical representation of OMS646 concentration (mean n=3 animals per group) versus time following OMS646 administration at the indicated dose. As shown in FIG. 13, at 5 mg/kg SC, the maximum plasma concentration of OMS646 of 5-6 μg/mL was reached approximately 1-2 days after dosing. The bioavailability of OMS646 at 5 mg/kg SC was approximately 60%. In FIG. 13 also shows that at 15 mg/kg SC, the maximum plasma concentration of OMS646 of 10-12 μg/mL was reached approximately 1-2 days after dosing. In all groups, OMS646 was slowly cleared from the systemic circulation with a terminal half-life of approximately 8-10 days. The profile of OMS646 was found to be typical of human antibodies in mice.

The PD activity of OMS646 is graphically shown in FIG. 14A and 14B. In FIG. 14A and 14B show the PD (decreased systemic lectin pathway) response for each mouse in the 5 mg/kg IV (FIG. 14A) and 5 mg/kg SC (FIG. 14B) groups. The dotted line indicates the baseline in the assay (maximum inhibition, intact mouse serum injected in vitro with an excess of OMS646 prior to assay). As shown in FIG. 14A, after IV administration of 5 mg/kg OMS646, the activity of the lectin pathway of the complement system immediately decreased to almost undetectable levels, and the recovery of lectin pathway activity during the 28-day observation period was moderate. As shown in FIG. 14B, mice dosed with 5 mg/kg of OMS646 SC exhibited a time-dependent suppression

- 70 043102 activity of the lectin pathway. The activity of the lectin pathway decreased to almost undetectable levels within 24 hours after drug administration and remained at a low level for at least 7 days. The activity of the lectin pathway increased gradually over time, but did not return to pre-dose levels during the 28 day follow-up period. The time-dependent lectin pathway activity profile observed after 15 mg/kg SC administration was similar to that for the 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. The data also indicate that doses of 5 mg/kg of OMS646 administered once a week, either IV or SC, are sufficient to achieve continuous downregulation of the activity of the lectin pathway of the complement system in mice.

Example 13

This example describes the production of recombinant antibodies that inhibit MASP-2 containing a heavy chain and/or light chain variable region containing one or more CDRs that specifically bind to MASP-2 and at least one SGMI nuclear peptide sequence (also called an anti-MASP-2 antibody carrying an SGMI peptide, or an antigen-binding fragment thereof).

Background/justification.

The design of specific MASP-2 inhibitors called 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 that was selected from a Schistocerca gregaria protease inhibitor 2 variants phage library in which six of the eight positions of the protease-binding loop were completely randomized. Subsequent in vitro evolution resulted in monospecific inhibitors with single digit KI values in the nM range (Heja et al., J. Biol. Chem., 287:20290, 2012). Structural studies have shown that the optimized protease binding loop forms a primary binding site that determines the specificity of the two inhibitors. The amino acid sequences of extended secondary and internal binding sites are common to both inhibitors and contribute to the contact site (Heja et al., 2012, J. Biol. Chem., 287:20290). Mechanically, SGMI-2 blocks activation of the complement lectin pathway without affecting the classical pathway (Heja et al., 2012, Proc. Natl. Acad. Sci., 109:10498).

The amino acid sequences of the SGMI-2 inhibitors are shown below.

SGMI-2L Full Size: LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (seq _GO NO: 72).

SGMI-2L, medium length: TCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (seq id NO: 73).

SGMI-2L, short: TCRCGSDGKSAVCTKLWCNQ (seq id NO: 74).

As described in this example, as well as in WO 2014/144542, anti-MASP-2 antibodies carrying the SGMI-2 peptide and fragments thereof were obtained by fusion with the amino acid sequence of the SGMI-2 peptide (SEQ ID NO: 72, 73 or 74) at the amino or carboxyl terminus of the heavy and/or light chain of a human anti-MASP-2 antibody. Ahtu-MASP-2 antibodies bearing the SGMI-2 peptide and fragments thereof have increased inhibitory activity compared to an exposed-backbone anti-MASP-2 antibody that does not contain the SGMI-2 peptide sequence as measured by C3b or C4b deposition assay. using human sera as described in WO 2014/144542 and also have increased inhibitory activity compared to naked-backbone anti-MASP-2 antibody measured in an in vivo mouse model. Methods for producing an anti-MASP-2 antibody bearing the SGMI-2 peptide are described below.

Methods.

Expression constructs were generated to encode four exemplary anti-MASP-2 antibodies bearing the SGMI-2 peptide, wherein the SGMI-2 peptide was fused to either the N- or C-terminus of the heavy or light chain of the representative MASP-2 inhibitory antibody OMS646 (as described in example 12).

Table 10

Fusion MASP-2 antibody/SGMI-2

Antibody comparisons Location of the peptide on the antibody SEQ ID NO: H-N n-s L-N L-C HL-M2 (naked MASP-2 OMS646) - - - - 67+70 H-M2-SGMI-2-N SGMI-2 - - - 75+70 H-M2-SGMI-2-C - SGMI-2 - - 76 +70 L-M2-SGMI-2-N - - SGMI-2 - 67+ 77 L-M2-SGMI-2-C - - - SGMI-2 67+ 78

Designations in the table. 10.

'Ή-N=amino-terminus of the heavy chain.

- 71 043102

H-C=carboxylic end of the heavy chain.

L-N=light chain amino terminus.

L-C'-carboxylic end of the light chain. M2 = backbone of MASP-2 ab (OMS646).

For N-terminal mergers shown in table. 10, a peptide linker ('GTGGGGGGSS' SEQ ID NO: 79) was added between the SGMI-2 peptide and the variable region.

For the C-terminal fusions 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 GSGA peptide (SEQ ID NO: 81) was added at the C-terminus of the fusion polypeptide to protect the C-terminal SGMI-2 peptides from degradation.

Amino acid sequences are shown below for the following representative MASP-2 antibody/SGMI-2 fusions.

H-M2ab6-SGMI-2-N (SEQ ID NO: 75, coded by SEQ ID NO: 82):

LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNOGTGGGSGSSSQVTLKESGPVLVK PTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFSSDEKSYRTSLKSRLTISKDTSK NQWLTMTNMDPVDTATYYCARIRRGGIDYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSES TA ALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[491 aa protein, aa l-36=SGMI-2 (underlined), aa 37-46=linker (italicized);

aa 47-164 = MASP-2 heavy chain variable region ab #6 (underlined);

aa 165-491=IgG4 constant region with mutation in the hinge region.]

H-M2ab6-SGMI-2-C (SEQ ID NO: 76, coded by SEQ ID NO: 83):

QVTLKESGPVLVKPTETLTLTTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFSSDEK SYRTSLKSRLTISKDTSKNQWLTMTNMDPVDTATYYCARIRRGGIDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKYPSDIAVEW ESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKAAGGSGLEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQGSGA

[491aa protein, aa 1-118=MASP-2 heavy chain variable region ab #6 (underlined);

aa 119-445=IgG4 constant region with mutation in the hinge region;

aa 446-451=first linker (in italics);

aa 452-487=SGMI-2;

aa 488-491=second linker (in italics).]

L-M2ab6-SGMI-2-N (SEQ ID NO: 77, encoded by SEQ ID NO: 84):

LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNOGTGGGSGSSSOPVL TOPPSLSVS PGQTASITCSGEKLGDKYAYWYOOKPGOSPVLVMYODKQRPSGIPERFSGSNSGNTATLTISGT OAMDEADYYCQAWDSSTAVFGGGTKLTVLGOPKAAPSVTLFPPSSEELQANKATLVCLIS D FYP GAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS

[258 aa protein, aa l-36=SGMI-2 (underlined);

aa 37-46=linker (in italics);

aa 47-152 = MASP-2 light chain variable region ab #6 (underlined);

aa 153-258 = constant region of human Ig lambda.]

L-M2ab6-SGMI-2-C (SEQ ID NO: 78, encoded by SEQ ID NO: 85):

OPVLTOPPSLSVSPGQTASITCSGEKL GDKYAYWYOQKPGQSPVLVMYQDKORPSGIPE RFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLGOPKAAPSVTL FP P S S E ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSY SCQVTHEGS TVE KTVAP TECS AAGGSGLEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLW CNQGSGA

[258 aa protein, aa 1-106 = MASP-2 light chain variable region ab #6 (underlined);

aa 107-212=human Ig lambda constant region;

- 72 043102 aa 213-218=first linker;

aa 219-254=SGMI-2;

aa 255-258=second linker.]

Functional analysis.

Four anti-MASP-2-SGMI-2 fusion antibody constructs were transiently expressed in Expi293F cells (Invitrogen), purified by protein A affinity chromatography, and tested in 10% normal human serum to suppress C3b deposition in a mannan-coated microsphere assay, as described below.

Testing of MASP-2-SGMI-2 fusions in a C3b deposition assay using mannan-coated microspheres.

MASP-2-SGMI-2 fusion antibodies were evaluated for their ability to inhibit the lectin pathway in a C3b deposition assay on mannan-coated microspheres. This assay, which allows determination of activity by flow cytometry, has higher resolution than the Wieslab® assay. Lectin pathway analysis using microspheres was performed as follows: Mannan was adsorbed onto 7 μM diameter polystyrene microspheres (Bangs Laboratories, Fishers, IN, USA) overnight at 4°C in carbonate-bicarbonate buffer (pH 9.6). The microspheres were washed with PBS and exposed to 10% human serum or 10% serum pre-incubated with antibodies or inhibitors. The serum-microsphere mixture was incubated at room temperature for 1 hour with stirring. After serum incubation, the microspheres were washed and C3b deposition on the microspheres was measured by detection with an anti-C3c polyclonal rabbit antibody (Dako North America, Carpinteria, CA, USA) and a PE-Cy5-conjugated goat anti-rabbit secondary antibody (Southern Biotech, Birmingham, AL, USA). After the staining procedure, the microspheres were analyzed using a FACSCalibur flow cytometer. Microspheres were analyzed as a homogeneous population using forward and side scatter and C3b deposition appeared as FL3 positive particles (FL-3 or FL-3 channel indicates the third or red channel on the cytometer). Population geometric mean fluorescence intensity (MFI) for each experimental condition was plotted against antibody/inhibitor concentration to assess lectin pathway inhibition.

IC ₅₀ values were calculated using GraphPad PRISM software. In particular, IC ₅₀ values were obtained by applying a non-linear (four parametric) sloped fit of log (antibody) versus mean fluorescence intensity curves obtained from cytometric analysis.

The results are shown in table. eleven.

Table 11

C3b deposition (mannan-coated microsphere assay) in 10% human serum

Design IC50 (NM) ab with naked N2 (mAb#6) > 3.63 nM H-M2-SGMI-2-N 2.11 nM L-M2-SGMI-2-C 1.99 pM H-M2-SGMI-2-N 2.24 pM L-M2-SGMI-2-N 3.71 pM

Results.

The control, SGMI-free anti-MASP-2 bare-backed antibody (mAb #6) was inhibitory in this assay with an IC ₅₀ value of >3.63 nM, consistent with the inhibition results observed in Example 12. Notably, , as shown in Table. 11, all SGMI-2-MASP-2 antibody fusions that were tested improved the performance of the MASP-2-backbone antibody in this assay, indicating that increased valence may also be useful in suppressing C3b deposition.

Testing MASP-2-SGMI-2 fusions in a mannan-coated microsphere assay to study C4b deposition with 10% human serum.

The C4b deposition assay was performed using 10% human serum under the same assay conditions as described above for the C3b deposition assay, with the following modifications. Analysis for C4b detection and flow cytometry analysis were performed by staining the deposition reaction with anti-C4b mouse monoclonal antibody (1:500, Quidel) and by staining with secondary goat anti-mouse F(ab') ₂ conjugated to PE-Cy5 (1:200, Southern Biotech), before flow cytometry analysis.

Results.

Both fusions SGMS-2-carrying anti-MASP-2-N-terminal antibody (H-M2-SGMI-2-N:

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IC ₅₀ = 0.34 nm, L-M2-SGMI-2-N: IC ₅₀ = 0.41 nM) had increased potency compared to aHTu-MASP-2-ocToe antibody (HL-M2: IC ₅₀ = 0, 78 nm).

Similarly, fusion of a single SGM-2-carrying C-terminal anti-MMS-2 antibody (H-M2-SGMI-2-C: IC ₅₀ =0.45 nM and L-M2-SGMI-2C: IC ₅₀ =0 .47 nM) had increased activity compared to the anti-MASP-2 backbone antibody (HL-M2: IC ₅₀ =1.2 nM).

MASP-2-SGMI-2 fusion testing in C3b deposition assay with mannan-coated microspheres with 10% mouse serum

C3b deposition assay with mannan-coated microspheres was performed as described above with 10% mouse serum. Similar to the results observed when using human sera, it was found that in mouse sera, fusions of SGMI-2-carrying anti-MASP-2 have increased efficiency compared to the anti-MASP-2 backbone antibody.

Brief review of the results. The results in this example demonstrate that all SGMI-2-MASP-2 antibody fusions that were tested improved the performance of the anti-MASP-2 backbone antibody.

Example 14

This example presents the results obtained using the unilateral ureteral obstruction (UUO) renal fibrosis model in MASP-2-/- deficient and MASP-2+/+ sufficient mice to assess the role of the lectin pathway in renal fibrosis.

Background/justification.

Kidney fibrosis and inflammation are characteristic features of advanced kidney disease. Tubulointerstitial fibrosis of the kidney is a progressive process involving persistent cell damage, aberrant healing, activation of resident and infiltrating kidney cells, release of cytokines, inflammation, and phenotypic activation of kidney cells to produce extracellular matrix. Tubulointerstitial (TI) renal fibrosis is a common endpoint for multiple kidney pathologies and represents a key target for potential therapies to prevent progressive renal dysfunction in chronic kidney disease (CKD). Renal TI failure is closely associated with decreased renal function in glomerular diseases (Risdon RA 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, in which myofibroblasts accumulate and the potential space between tubules and peritubular capillaries becomes clogged with a matrix composed of collagens and other proteoglycans. The origin of myofibroblast TI remains highly controversial, but fibrosis is usually preceded by inflammation, which is initially characterized by TI accumulation of T-lymphocytes and then macrophages (Liu Y. et al., Nat. Rev. Nephrol., 7:684-696, 2011; Duffield J.S., J. Clin. Invest., 124:2299-2306, 2014).

The rodent UUO model generates progressive renal fibrosis, the hallmark of progressive kidney disease of virtually any etiology (Chevalier et al., Kidney International, 75:1145-1152, 2009). There are reports of increased C3 gene expression in wild-type mice after UUO and a significant decrease in collagen deposits in C3-/- knockout mice after UUO compared to wild-type mice, suggesting a role for complement activation in renal fibrosis (Fearn et al., Mol. Immunol., 48:1666-1733, 2011). There is also a report that C5 deficiency resulted in significant attenuation of major components of renal fibrosis in a tubulointerstitial lesion model (Boor P. et al., J. of Am. Soc. of Nephrology, 18:1508-1515, 2007). However, prior to the study described herein by the inventors, the specific complement components involved in renal fibrosis were not well defined. Therefore, the following study was conducted to evaluate MASP-2 (-/-) and MASP-2 (+/+) mice in a unilateral ureteral obstruction (UUO) model.

Methods.

Received MASP-2 -/- mouse, as described in example 1, and subjected to backcrossing for 10 generations with C57BL/6. Wild-type (WT) C57BL/6 mice and homozygous MASP-2-deficient (MASP-2-/-) mice in the background of C57BL/6 were maintained under standardized conditions with a 12/12 day/night cycle, while mice were fed standard food pellets , water and food were freely available. Ten-week-old mice, 6 per group, were anesthetized with 2.5% isoflurane in 1.5 L/min of oxygen. The right ureters in two groups of ten week old male C56/BL6 mice, wild type and MASP-2 -/- were surgically ligated. The right kidney was exposed through a 1 cm lateral incision. The right ureter was completely blocked at two points using a 6/0 polyglactin suture. Anesthesia was performed with buprenorphine intraoperatively every 12 hours for up to 5 doses depending on the degree of pain. Local anesthesia with bupivacaine was performed once during the operation.

Mice were sacrificed 7 days after surgery, and kidney tissues were collected, fixed and embedded in paraffin blocks. Blood was collected from mice by cardiac puncture under anesthesia, and mice were bled after nephrectomy. The blood was left on ice to coagulate for 2 hours and the serum was separated by centrifugation and stored frozen in aliquots at -80°C.

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Immunohistochemistry of kidney tissue.

To measure the degree of fibrosis in the kidneys by collagen deposition, 5 µm paraffin sections of the kidneys were stained with picrosirius red, collagen-specific stains, as described in Whittaker P. et al., Basic. Res. Cardiol., 89:397-410, 1994. Briefly, kidney sections were deparaffinized, rehydrated, and collagen stained for 1 h with an aqueous solution of picrosirius red (0.5 g Sirius red, Sigma, Dorset UK) in 500 ml of saturated aqueous picric acid. . Slides were washed twice with acidified water (0.5% glacial acetic acid in distilled water) for 5 minutes each time, then dehydrated and prepared for study.

To measure the degree of inflammation as evidenced by macrophage infiltration, kidney sections were stained with F4/80 macrophage-specific antibody as follows. Formalin-fixed, paraffin-embedded, 5-micron kidney sections were deparaffinized and rehydrated. Antigen unmasking was performed in citrate buffer at 95°C for 20 min, followed by blocking of endogenous peroxidase activity by incubation in 3% H ₂ O ₂ for 10 min. 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 blocking with avidin/biotin. Tissue sections were washed with PBS after each step three times for 5 min. Anti-macrophage F4/80 primary antibody (Santa Cruz, Dallas, TX, USA) diluted 1:100 in blocking buffer was applied for 1 h. Then biotinylated goat anti-rat secondary antibody diluted 1:200 was applied for 30 min. followed by application of horseradish peroxidase (HRP) conjugated enzyme for 30 min. The color was developed with diaminobenzidine (DAB) substrate (Vector Labs, Peterborough UK) for 10 min, the slides were washed in water, dehydrated and prepared for examination without counterstaining for ease of computer analysis.

Image analysis.

The percentage staining of the cortical layer of the kidneys was determined as described by Furness P.N. et al., J. Clin. Pathol., 50:118-122, 1997. Briefly, 24-bit color images were taken from consecutive non-overlapping fields of the renal cortex just below the renal capsule around the entire periphery of the kidney slice. After each image capture, the NIH image was used to extract the red channel from it as an 8-bit monochrome image. Background illumination unevenness was subtracted using a pre-recorded image of the illuminated microscope field without a slice placed on it. The image was given a fixed threshold to determine the area of the image corresponding to positive coloration. Then, the percentage of black pixels was calculated, and after all the images around the kidney were measured in this way, the average percentage was recorded, providing a value corresponding to the percentage of the stained area in the kidney section.

Analysis of gene expression.

Expression of several genes related to kidney inflammation and fibrosis in mouse kidney was measured by quantitative PCR (qPCR) as follows. Total RNA was isolated from the renal cortex using Trizol® (ThermoFisher Scientific, Paisley, UK) according to the manufacturer's instructions. The extracted RNA was processed with a DNA-free Turbo kit (ThermoFisher Scientific) to eliminate DNA contamination, and then the first strand of cDNA was synthesized using an AMV reverse transcription system (Promega, Madison, WI, USA). cDNA integrity was confirmed by a single qPCR reaction using the TaqMan GAPDH Assay (Applied Biosystems, Paisley UK) followed by a qPCR reaction using 96-well Custom TaqMan Array plates (Life Technologies, Paisley, UK).

Twelve genes were examined in this analysis:

collagen type IV alpha 1 (col4a1; assay ID: Mm01210125_m1);

transforming growth factor beta-1 (TGFe-1; assay ID: Mm01178820_m1);

caderin 1 (Cdh1; assay ID: Mm01247357_m1);

fibronectin 1 (Fn1; assay ID: Mm01256744_m1);

interleukin 6 (IL6; Assay ID Mm00446191_ml);

interleukin 10 (IL10; Assay ID Mm00439614_m1);

interleukin 12a (IL12a; Assay ID Mm00434165_m1);

vimentin (Vim; assay ID Mm01333430_m1);

actinin alpha 1 (Actn1; assay ID Mm01304398_m1);

tumor necrosis factor-α (TNF-α, assay ID Mm00443260_g1);

complement component 3 (C3; assay ID Mm00437838_m1);

interferon-gamma (Ifn-γ; Assay ID Mm01168134).

The following constitutive control genes were used:

glyceraldehyde-3-phosphate dehydrogenase (GAPDH, assay ID Mm99999915_g1);

glucuronidase beta (Guse; Assay ID Mm00446953_m1);

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hypoxanthine guanine phosphoribosyltransferase (HPRT; Assay ID Mm00446968_m1).

µl of reactants were amplified using TaqMan Fast Universal Master Mix (Applied Biosystems) for 40 cycles. Real time PCR amplification data was analyzed using Applied Biosystems 7000 SDS v1.4 software.

Results.

Following unilateral ureteral obstruction (UUO), the occluded kidneys show an influx of inflammatory cells, particularly macrophages, followed by a rapid development of fibrosis, as evidenced by collagen accumulation along with tubular dilatation and thinning of the proximal tubular epithelium (see Chevalier R.L. et al., Kidney Int. ., 75:1145-1152, 2009).

In FIG. 15 graphically shows the results of computer analysis of images of Sirius red stained kidney tissue sections where tissue sections were obtained from wild-type and MASP-2-/ mice 7 days after ureteral obstruction (UUO) and from sham-operated control mice. As shown in FIG. 15, kidney sections of wild-type mice 7 days after ureteral obstruction showed significantly more collagen deposits compared to MASP-2-/ mice (p value=0.0096). The mean values ± standard error of the mean for UUO operated mice in the wild-type and MASP-2 -/- groups were 24.79±1.908 (n=6) and 16.58+1.3 (n=6), respectively. As further shown in FIG. 15, tissue sections from sham-operated wild-type control mice and sham-operated control MASP-2 −/− mice showed very low levels of collagen staining, as expected.

In FIG. 16 graphically shows the results of computer analysis of images of kidney tissue sections stained with antibodies specific for macrophage marker F4/80, where tissue sections were obtained from wild-type and MASP-2 -/- mice 7 days after ureteral obstruction or from sham-operated control mice. As shown in FIG. 16, compared with WT mice, tissues obtained from UUO kidneys of MASP-2-/- mice showed significantly less macrophage infiltration 7 days after ureteral obstruction (% area of macrophages stained in WT: 2.23 ± 0.4 vs. MASP-2-/-: 0.53±0.06, p=0.0035). As further shown in FIG. 16, tissue sections from sham-operated wild-type mice and sham-operated MASP-2 −/− mice showed no detectable macrophage staining.

Expression analysis of many different genes associated with kidney inflammation and fibrosis was performed on kidney tissue sections obtained from wild-type and MASP-2-/- mice 7 days after ureteral obstruction and from sham-operated wild-type and MASP-2-/ mice. - mice. The data shown in FIG. 17-20 are Log ₁₀ relative numbers in a sample of sham-operated wild-type mice, and bars represent the standard error of the mean. With regard to the results of the analysis of the expression of genes associated with fibrosis, in Fig. 17 graphically shows the relative expression levels of collagen type IV alpha 1 (collagen-4) mRNA measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 -/- mice 7 days after ureteral obstruction and from sham-operated control mice. . In FIG. 18 is a graphical representation of the relative expression levels of transforming growth factor beta-1 (TGFe-1) mRNA measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 −/− mice 7 days after ureteral obstruction and from sham-operated controls. mice. As shown in FIG. 17 and 18, the occluded kidneys of wild-type mice showed significantly increased expression of the fibrosis-associated genes collagen type IV (Fig. 17) and TGFe-1 (Fig. 18) compared to sham-operated kidneys in wild-type mice, indicating that these Fibrosis-related genes were induced after UUO injury in wild-type mice, as expected. On the contrary, as further shown in FIG. 17 and 18, the occluded kidneys of MASP-2 −/− mice subjected to UUO injury showed a significant decrease in type IV collagen expression (Fig. 17, p=0.0388) and a significant decrease in TGFe-1 expression (Fig. 18, p= 0.0174) compared to wild-type mice subjected to UUO damage.

With regard to the results of the analysis of the expression of genes associated with inflammation, in Fig. 19 graphically shows the relative levels of interleukin-6 (IL-6) mRNA expression measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 -/- mice 7 days after ureteral obstruction and from sham-operated control mice. In FIG. 20 graphically shows the relative levels of interferon-y mRNA expression as measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2 -/- mice 7 days after ureteral obstruction and from sham-operated control mice. As shown in FIG. 19 and 20, occluded kidneys from wild-type mice showed significantly increased expression of the inflammation-associated genes interleukin-6 (Fig. 19) and interferon-γ (Fig. 20) compared to sham-operated kidneys from wild-type mice, indicating that these inflammation-related genes are induced after UUO injury in wild-type mice. On the contrary, as further shown in FIG. 19 and 20, the occluded kidneys of MASP-2 −/− mice subjected to UUO injury showed a significant decrease in the expression of interleukin-6 (Fig. 19, p=0.0109) and interferon-γ (Fig. 20, p =0.0182) compared to wild-type mice subjected to UUO lesion.

Of note, expression of the Vim, Actn-1, TNFa, C3, and IL-10 genes is significantly upregulated in UUO kidneys from both wild-type and MASP-2-/- mice, with no significant difference in the expression levels of these particular mice. genes between wild-type and MASP-2 −/− mice (data not shown). The expression levels of the Cdh-1 and IL-12a genes did not change in the occluded kidneys of animals in any of the groups (data not shown).

Discussion.

The rodent UUO model is recognized to induce early, active, and deep injury in the occluded kidney with reduced renal blood flow, interstitial inflammation, and rapid fibrosis within one to two weeks of obstruction and has been widely used to understand the general mechanisms and mediators of inflammation and fibrosis in the kidney ( see, for example, 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 compared to control (+/+) wild-type mice. Surprising results indicating a significant reduction in kidney damage both at the histological level and at the level of gene expression in MASP-2-/- animals show that the lectin pathway of complement activation has a significant effect on the development of inflammation and fibrosis in the occluded kidney. Without wishing to be bound by a particular theory, it is believed that the lectin pathway has a significant impact on the pathophysiology of fibrotic disease by triggering and maintaining pro-inflammatory stimuli that perpetuate a vicious cycle in which cell damage causes inflammation, which in turn causes further cell damage, scarring, and loss. fabrics. Based on these results, suppression or blockade of MASP-2 by an inhibitor is expected to have a prophylactic and/or therapeutic effect, manifested in the suppression or prevention of renal fibrosis, as well as the suppression or prevention of fibrosis in general (i.e., regardless of tissue or organ ).

Example 15

This example describes an analysis of the efficacy of a MASP-2 monoclonal inhibitory antibody in a unilateral ureteral obstruction (UUO) model, a mouse model of renal fibrosis.

Background/justification.

Relief of tubulointerstitial fibrosis, a common endpoint of many renal pathologies, is an important target for therapies aimed at preventing the progression of renal disease. Given the paucity of new and existing therapies targeting the inflammatory profibrotic pathways in kidney disease, there is an urgent need to develop new therapies. Many patients with proteinuric kidney disease have tubulointerstitial inflammation and progressive fibrosis, which are closely associated with decreased kidney function. Proteinuria itself causes tubulointerstitial inflammation and leads to the development of proteinuric nephropathy (Brunskill N.J. et al., J. Am. Soc. Nephrol., 15:504-505, 2004). Regardless of primary kidney disease, tubulointerstitial inflammation and fibrosis are invariably observed in patients with progressive renal failure and are closely correlated with decreased excretory function (Risdon RA et al., Lancet, 1:363-366, 1968; Schainuck L.I. et al., Hum. Pathol ., 1:631640, 1970). Therapies that can interrupt common key cellular pathways leading to fibrosis will find widespread use in renal disorders.

As described in Example 14, in a UUO model of non-proteinuric renal fibrosis, MASP-2 -/- mice were found to have significantly lower levels of kidney fibrosis and inflammation compared to wild-type controls, as evidenced by inflammatory cellular infiltrates (75% ) and histological markers of fibrosis such as collagen (a one-third decrease), thus proving the key role of the lectin pathway in renal fibrosis.

As described in Example 13, the synthesized anti-MASP-2 monoclonal antibody (OMS646-SGMI-2 fusion protein containing the SGMI-2 peptide fused to the C-terminus of the OMS646 heavy chain) was also shown to specifically block lectin pathway function in humans , blocks the lectin pathway in mice. In this example, OMS646-SGMI-2 was analyzed in a wild type mouse UUO kidney fibrosis model to determine the ability of a specific MASP-2 inhibitor to suppress renal fibrosis.

Methods.

This study evaluated the effect of a MASP-2 inhibitory antibody (10 mg/kg OMS646-SGMI-2) versus an anti-human IgG4 isotype antibody (10 mg/kg ET904) and a delivery medium control in male WT C57BL/6 mice. . Antibodies (10 mg/kg) were administered to groups of 9 mice by intraperitoneal (ip) injection on days 7, 4 and 1 before UUO surgery and then on day 2 after surgery. Blood samples were taken before the administration of antibodies, and at the end of the experiment, the functional activity of the lectin pathway was evaluated.

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UUO operation, tissue harvesting, and staining with Sirius Red and F4/80 macrophage-specific antibody were performed using the methods described in Example 14.

The content of hydroxyproline in the kidneys of mice was measured using a specific test kit for colorimetric analysis (Sigma) according to the manufacturer's instructions.

To assess the pharmacodynamic effect of inhibitory mAb-MASP-2 in mice, the activity of the lectin pathway of the complement system was assessed by quantifying lectin-induced C3 activation in minimally diluted serum samples collected at the indicated time after ip administration of MASP-2 mAb or control mAb to mice. Briefly, 7 μM diameter polystyrene microspheres (Bangs Laboratories, Fisher IN, USA) were coated with mannan by incubating overnight with 30 μg/ml mannan (Sigma) in sodium bicarbonate buffer (pH 9.6), then washed, blocked with 1% fetal bovine serum in PBS and resuspended in PBS to a final concentration of 1x10 ⁸ mirospheres/ml. Complement deposition reactions were initiated by adding 2.5 µl of mannan-coated microspheres (~250,000 microspheres) to 50 µl of minimally diluted mouse serum samples (final serum concentration 90%) followed by incubation for 40 min at 4°C. After stopping the deposition reaction by adding 250 µl of ice-cold flow cytometry buffer (FB:PBS containing 0.1% fetal bovine serum), the microspheres were collected by centrifugation and washed two more times with 300 µl of ice-cold FB.

To quantify lectin-induced C3 activation, microspheres were incubated for 1 h at 4°C with 50 μl of rabbit anti-human C3c antibody (Dako, Carpenteria, CA, USA) diluted in FB. After two washes with FB to remove unbound material, the microspheres were incubated for 30 min at 4°C with 50 μl of PECy5-conjugated goat anti-rabbit antibody (Southern Biotech, Birmingham, AL, USA) diluted in FB. After two washes with FB to remove unbound material, the microspheres were resuspended in FB and analyzed using a FACS Calibur cytometer. The microspheres were analyzed as a homogeneous population using forward and side scatter and the C3b deposition in each sample was quantified as mean fluorescence intensity.

Results.

Assessment of collagen deposition.

In FIG. 21 is a graphical representation of the results of computerized image analysis of Sirius Red-stained renal tissue sections, where the tissue sections were obtained 7 days after ureteral obstruction in wild-type mice treated with either a MASP-2 inhibitory antibody or an isotype control antibody. As shown in FIG. 21, tissue sections from kidneys harvested 7 days post-obstruction (UUO) from wild-type mice treated with an inhibitory MASP-2 antibody showed a significant reduction (p=0.0477) in collagen deposition compared to the amount of collagen deposition in the sections. tissues from occluded kidneys obtained from wild-type mice treated with IgG4 isotype control.

Assessment of hydroxyproline content.

Hydroxyproline was measured in kidney tissues as an indicator of collagen content. Hydroxyproline is a parameter that is a very indicative indicator of the pathophysiological progression of the disease induced in this model.

In FIG. 22 is a graphical representation of the hydroxyproline content in kidneys collected 7 days post-obstruction (UUO) from wild-type mice treated with either a MASP-2 inhibitory antibody or an isotype control antibody. As shown in FIG. 22, occluded kidney tissues from mice treated with MASP-2 inhibitory antibody showed a significant decrease in hydroxyproline, an indicator of collagen content, compared to kidneys from mice treated with the isotype control mAb, IgG4 (p=0.0439).

Assessment of inflammation.

In occluded kidneys from wild-type animals treated with isotype control antibody and wild-type animals treated with MASP-2 inhibitory antibody, an abundant macrophage infiltrate was observed. Careful quantification revealed no significant difference in percentage of stained macrophages between the two groups (data not shown). However, despite an equivalent number of infiltrating macrophages in occluded kidneys from animals injected with inhibitory MASP-2 antibodies, fibrosis developed to a significantly lesser extent, as judged by Sirius red staining, compared with occluded kidneys from animals injected with isotype control, this result is consistent with results that occluded kidney tissues from mice treated with an inhibitory MASP-2 antibody had significantly less hydroxyproline than kidneys from mice treated with the isotype control mAb, IgG4.

Discussion.

The results described in this example showed that the use of a MASP-2 inhibitory antibody provides protection against kidney fibrosis in a UUO model, which is consistent with the results described in example 14 demonstrating that kidney fibrosis and inflammation in MASP-2 -/- mice in UUO models appeared to a significantly lesser extent than wild-type mice. The results in this example demonstrate reduced fibrosis in mice treated with a MASP-2 inhibitory antibody. The finding that fibrosis in UUO kidneys is reduced in animals with a reduction or blockade of MASP-2-dependent lectin pathway activity is a very significant new finding. Taken together, the results presented in Example 14 and this example demonstrate the beneficial effect of MASP-2 inhibition on renal tubulointerstitial inflammation, tubular cell damage, profibrotic cytokine release, and scarring. The reduction of renal fibrosis remains a key goal of kidney treatment. The UUO model is a severe condition model with accelerated renal fibrosis, and an intervention that reduces fibrosis in this model, such as the use of MASP-2 inhibitory antibodies, is likely to be used to suppress or prevent renal fibrosis. The results of the UUO model can probably also be attributed to kidney diseases characterized by glomerular and/or proteinuric tubular damage.

Example 16

This example presents the results that were obtained using a model of fibrosis, inflammation and tubulointerstitial injury in the background of proteinuria caused by overdose of proteinuria in MASP-2 -/- mice and wild-type mice to evaluate the role of the lectin pathway in proteinuric nephropathy.

Background/justification.

Proteinuria is a risk factor for renal fibrosis and loss of excretory function independent of primary kidney disease (Tryggvason K. et al., J. Intern. Med., 254:216-224, 2003; Williams M., Am. J. Nephrol., 25:77-94, 2005). The concept of proteinuric nephropathy describes the toxic effects of excess protein entering the proximal tubules as a result of impaired glomerular selective 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 in many glomerular diseases, leads to a proinflammatory scarring environment in the kidney and is characterized by changes in proximal tubular cell growth, apoptosis, gene transcription, and inflammatory cytokine production as a consequence of deregulated signaling pathways stimulated by proteinuric tubular fluid. Proteinuric nephropathy is generally recognized as a key factor contributing to the progressive kidney damage that is characteristic of various primary kidney pathologies.

Chronic kidney disease affects more than 15% of the adult population in the United States and is responsible for approximately 750,000 deaths worldwide each year (Lozano R. et al., Lancet, vol. 380, Issue, 9859:2095-2128, 2012). Proteinuria is an indicator of chronic kidney disease and a contributing factor to disease progression. Many patients with proteinuric kidney disease have tubulointerstitial inflammation and progressive fibrosis, which are closely associated with decreased kidney function. Proteinuria itself causes tubulointerstitial inflammation and leads to the development of proteinuric nephropathy (Brunskill N.J. et al., J. Am. Soc. Nephrol., 15:504-505, 2004). In proteinuric kidney disease, excessive amounts of albumin and other macromolecules are filtered through the glomeruli and reabsorbed by proximal tubular epithelial cells. This induces a vicious inflammatory cycle mediated by complement activation leading to cytokine and leukocyte infiltrates that cause interstitial tubular injury and fibrosis, thereby exacerbating proteinuria and leading to loss of kidney function and eventually progression to end-stage renal disease ( see, for example, Clark et al., Canadian Medical Association Journal, 178:173-175, 2008). Therapies that modulate this harmful cycle of inflammation and proteinuria are expected to improve outcome in chronic kidney disease.

Considering the positive effect of MASP-2 inhibition in the UUO tubulointerstitial injury model, the following experiment was performed to determine if MASP-2 inhibition could reduce kidney injury in the protein overdose model. This study used protein overdose to induce proteinuric kidney disease as described in Ishola et al., European Renal Association, 21:591-597, 2006.

Methods.

Received MASP-2 -/- mouse, as described in example 1, and subjected to backcrossing for 10 generations with BALB/c. The present study compared the results obtained with wild-type mice and MASP-2-/- BALB/c mice in a model of proteinuria induced by overdose.

One week prior to the start of the experiment, mice had one kidney removed prior to protein overdose in order to obtain an optimal response. The proteinuria-inducing agent was low endotoxin bovine serum albumin (BSA, Sigma) administered in normal saline to WT (n=7) and MASP-2-/- mice (n=7) at the following doses: one dose per 2 mg BSA/g, 4 mg BSA/g, 6 mg BSA/g, 8 mg BSA/g, 10 mg BSA/g and 12 mg BSA/g bw and 9 doses of 15 mg BSA/g bw, in i.p. was administered over a total of 15 days. 15 doses. Control mice WT (n=4) and

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MASP-2-/- (n=4) mice received saline administered only intraperitoneally. After the last dose, the animals were housed singly in metabolic cages for 24 hours to collect urine. Blood was collected by cardiac puncture under anesthesia, the blood was left to coagulate on ice for 2 hours, and the 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 injection on day 15, and various tissues were collected for analysis. Kidneys were harvested and processed for H&E staining and immunostaining. Immunohistochemical staining was performed as follows. Formalin-fixed, paraffin-embedded, 5-micron sections of kidney tissue from each mouse were deparaffinized and rehydrated. Antigen unmasking was performed _in citrate buffer at 95°C for 20 min followed by tissue incubation in 3% _H2O2 for 10 min. The tissues were then incubated in blocking buffer (10% serum from the species in which the secondary antibody was grown and 1% BSA in PBS) with 10% avidin for 1 h at room temperature. Tissue sections were washed with PBS after each step three times for 5 min. Then the primary antibody was used in blocking buffer with 10% biotin solution for 1 hour at a concentration of 1:100 for antibodies F4/80 (Santa Cruz, cat. no. sc-25830), TGFe (Santa Cruz, cat. 1 sc-7892) , IL-6 (Santa Cruz, cat. 1 sc-1265) and a concentration of 1:50 for the TNFa antibody (Santa Cruz, cat. 1 sc-1348). Biotinylated secondary antibody was then applied for 30 min at a concentration of 1:200 for F4/80, TGFe and IL-6 sections and 1:100 for TNFa sections, followed by application of the HRP enzyme conjugate for another 30 min. Staining was performed with a diaminobenzidine (DAB) substrate kit (Vector Labs) for 10 min and the slides were washed in water, dehydrated and prepared for examination without counterstaining to simplify computer analysis. Stained tissue sections from the renal cortex were analyzed by digital image capture followed by quantification using automatic image analysis software.

Apoptosis was assessed in tissue sections by staining the dUTP end label using terminal deoxynucleotidyl transferase (TUNEL) as follows. Apoptotic cells in kidney sections were stained using the ApopTag® Peroxidase Kit (Millipore) as follows. Paraffin-embedded, formalin-fixed kidney sections from each mouse were deparaffinized, rehydrated, and then the protein was permibilized with proteinase K (20 μg/ml), which was applied to each sample for 15 min at room temperature. Between stages, the samples were washed with PBS. Endogenous peroxidase activity was quenched by tissue incubation in 3% H ₂ O ₂ for 10 min. The tissues were then incubated in equilibration buffer followed by incubation with TdT enzyme for 1 h at 37°C. After washing in stop/wash buffer for 10 minutes, the anti-digoxigenin conjugate was applied for 30 minutes at room temperature, followed by washing. Staining was carried out using a kit with DAB-substrate for 4 min, followed by washing in water. The tissues were counterstained in hematoxylin and mounted in DBX. The frequency of TUNEL-stained (brown) apoptotic cells was manually counted in sequentially selected 20 high magnification cortical fields using a Leica DBXM light microscope.

Results.

Assessment of proteinuria.

To confirm the presence of proteinuria in mice, total serum protein was analyzed on day 15, and total protein excreted was measured in urine samples collected over 24 hours on day 15 of the study.

In FIG. 23 is a graphical representation of total serum proteins (mg/mL) measured at day 15 in WT mice (n=2) treated with saline only, WT mice treated with BSA (n=6), and MASP- 2-/- mice treated with BSA (n=6). As shown in FIG. 23, BSA administration increased the total serum protein level in both the wild-type and MASP-2-/- groups more than twice the concentration in the saline-only control group, with between groups receiving treatment, no significant difference was observed.

In FIG. 24 is a graphical representation of the total amount of protein (mg) excreted in urine collected over 24 hours on day 15 of the study from WT (n=2) mice treated with saline only, WT mice treated with BSA (n= 6) and MASP-2 -/- mice treated with BSA (n=6). As shown in FIG. 24, on day 15 of this study, an approximately six-fold increase in total urinary protein excreted in the BSA treated groups compared to the sham-treated control group that received only saline was observed. The results shown in FIG. 23 and 24 indicate that the proteinuria model works as expected.

Evaluation of histological changes in the kidneys.

In FIG. 25 shows representative H&E-stained kidney tissue sections that were collected on

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Day 15 protein overdose study from mice of the following groups:

wild-type control mice (panel A);

MASP-2-/- control mice (panel B);

wild-type mice treated with BSA (panel C); and (panel D) MASP-2 -/- mice treated with BSA (panel D).

As shown in FIG. 25, the MASP-2-/- overdose group (panel D) showed significantly higher tissue preservation compared to the wild-type overdose group (panel C) at the same level of protein overdose challenge. For example, Bowman's capsules in wild-type mice treated with BSA (overdose) (panel C) were significantly expanded compared to Bowman's capsules in the control wild-type group (panel A). In contrast, Bowman's capsules of MASP-2-/(overdose) mice treated with the same amount of BSA (panel D) maintained a morphology similar to that observed in control MASP-2-/- mice (panel B) and wild-type control mice. type (panel A). As further shown in FIG. 25, large cast structures of proteins accumulate in the proximal and distal tubules of the renal sites in wild-type (panel C), which are larger and more numerous than those in MASP-2 -/- mice (panel D).

It should also be noted that analysis of kidney sections in this study using a transmission electron microscope showed that mice treated with BSA had a general lesion at the ciliary margins in the distal and proximal cells of the renal tubule with cell content and nuclei embedded in the tubular lumen. In contrast, tissue in MASP-2 −/− mice treated with BSA was preserved.

Assessment of macrophage infiltration in the kidneys.

To measure the degree of inflammation, as evidenced by macrophage infiltration, tissue sections of harvested kidneys were also stained with an antibody to the F4/80 macrofan marker using the methods described in Boor et al., J. of Am. soc. Nephrology, 18:1508-1515, 2007.

In FIG. 26 graphically shows the results of computer analysis of images of kidney tissue sections stained with F4/80 macrophage marker antibody showing the mean macrophage area stained (%) where the tissue sections were obtained on day 15 of protein overdose study in wild-type control mice (n=2 ), wild-type mice treated with BSA (n=6), and MASP-2 -/- mice treated with BSA (n=5). As shown in FIG. 26, sections of kidney tissue stained with F4/80 macrophage marker antibody indicate that although both BSA-treated groups showed a significant increase in macrophage infiltration in the kidney (measured as % area of F4/80 antibody stained) compared to In sham-treated control wild-type mice, a significant reduction in macrophage infiltration was observed in tissue sections of BSA-treated MASP-2 −/− mice compared to macrophage infiltration in tissue sections of BSA-treated wild-type mice (p value = 0.0345).

In FIG. 27A is a graphical analysis of the presence of a correlation between macrophage levels and proteinuria in each BSA-treated wild-type mouse (n=6) as a plot of total protein excreted, measured in urine from a 24-hour sample, versus degree of macrophage infiltration (% mean painted area). As shown in FIG. 27A, most wild-type kidney samples showed a positive correlation between the level of proteinuria and the degree of macrophage infiltration.

In FIG. 27B is a graphical analysis of the presence of a correlation between macrophage levels and proteinuria in each BSA-treated MASP-2-/- mice (n=5) as a plot of total protein excreted measured in urine from a 24-hour sample against degree of infiltration. macrophages (% of mean stained area). As shown in FIG. 27B, the positive correlation observed in wild-type mice between the level of proteinuria and the degree of macrophage infiltration (shown in FIG. 27A) was not observed in MASP-2 -/- mice. While not wishing to be bound by a particular theory, these results may indicate a clearing mechanism for inflammation at high levels of proteinuria in MASP-2 -/- mice.

Assessment of cytokine infiltration.

Interleukin 6 (IL-6), transforming growth factor beta (TGFe), and tumor necrosis factor alpha (TNFa) are pro-inflammatory cytokines known to be activated in the proximal tubules of wild-type mice in a proteinuria model (Abbate M. et al.. Journal of the American Society of Nephrology: JASN, 17:2974-2984, 2006 David S. et al., Nephrology, Didalysis, Transplantation, Official Publication of the European Dialysis and Transplant Association - European Renal Association, 12:51-56, 1997). Kidney tissue sections were stained with cytokine-specific antibodies as described above.

In FIG. 28 graphically shows the results of computer analysis of images of tissue sections stained with anti-TGFe antibody (measured as % area of anti-TGFe antibody stained) 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 TGFe staining was observed in the BSA (overdose) group of wild-type mice compared to the BSA (overdose) group of MASP-2 -/- mice (p=0.026).

In FIG. 29 graphically shows the results of computer analysis of images of tissue sections stained with anti-TNFa antibody (measured as % area of anti-TNFa antibody stained) in wild-type mice treated with BSA and MASP-2-/- mice treated with BSA (n= 5). As shown in FIG. 29, a significant increase in TNFa staining was observed in the wild-type group treated with BSA (overdose) compared to the MASP-2-/- group treated with BSA (overdose) (p=0.0303).

In FIG. 30 graphically shows the results of computer analysis of images of tissue sections stained with anti-III-6 antibody (measured as % area of anti-HE-6 antibodies stained) in control wild-type mice, control MASP-2-/- mice, wild-type mice, treated with BSA (n=7), and MASP-2 -/- mice treated with BSA (n=7). As shown in FIG. 30, a very significant increase in IL-6 staining was observed in the BSA-treated wild-type group compared to the MASP-2-/BSA-treated group (p=0.0016).

Apoptosis assessment.

Apoptosis was assessed in tissue sections by staining the dUTP end-mark using terminal deoxynucleotidyl transferase (TUNEL), and the frequency of TUNEL-stained apoptotic cells was counted in sequentially selected 20 high magnification cortical fields (HPF).

In FIG. 31 graphically shows the frequency of TUNEL-positive apoptotic cells counted in sequentially selected 20 high magnification fields of view (HPF) in tissue sections of the renal cortex in wild-type control mice (n=1), control MASP-2 -/- mice (n =1), wild-type mice treated with BSA (n=6), and MASP-2 -/- mice treated with BSA (n=7). As shown in FIG. 31, a significantly higher level of apoptosis in the cortex was observed in kidneys obtained from wild-type mice treated with BSA compared to kidneys obtained from MASP-2 -/- mice treated with BSA (p=0.0001).

General review of results and conclusions.

The results of this example indicate that the MASP-2 -/- mouse has reduced kidney damage in a protein overdose model. Therefore, MASP-2 inhibitory agents, such as MASP-2 inhibitory antibodies, are expected to suppress or prevent the deleterious cycle of inflammation and proteinuria and improve outcomes in chronic kidney disease.

Example 17.

This example describes the analysis of monoclonal inhibitory antibody MASP-2 for efficacy in reducing and/or preventing inflammation of the kidneys and tubulointerstitial damage in a model of proteinuria caused by overdose of proteinuria in wild-type mice.

Background/justification.

As described in Example 16, in a protein-induced proteinuria model, MASP-2 -/- mice were found to show significantly better outcomes (eg, less tubulointerstitial injury and less kidney inflammation) than wild-type mice, implying a pathogenic role. lectin pathway in proteinuric kidney disease.

As described in Example 13, a MASP-2 inhibitory monoclonal antibody (OMS646-SGMI-2) was generated that specifically blocks the function of the lectin pathway in humans and has also been shown to block the lectin pathway in mice. In this example, the efficacy of the MASP-2 inhibitory antibody OMS646-SGMI-2 was evaluated in a mouse model of proteinuria overdose to reduce and/or prevent kidney inflammation and tubulointerstitial injury in wild-type mice.

Methods.

This study evaluated the effect of a MASP-2 inhibitory antibody (10 mg/kg OMS646-SGMI-2) versus an anti-human IgG4 isotype control antibody (10 mg/kg ET904) and a saline control.

Similar to the study described in Example 16, this study used protein overdose to induce proteinuric kidney disease (Ishola et al., European Renal Association, 21:591-597, 2006). Proteinuria was induced in Balb/c mice with one kidney removed daily i.p. injections of escalating doses (2 to 15 g/kg) of low endotoxin bovine serum albumin (BSA) for 15 days as described in Example 16.

Antibody treatment was carried out by i.p. injections twice a week starting 7 days before the induction of proteinuria throughout the study. This dosing regimen was chosen based on previous PK/PD and pharmacological studies demonstrating sustained suppression of the lectin pathway (data not shown). Mice were sacrificed on day 15, kidneys were harvested and processed for H&E staining and immunostaining. Stained tissue sections from the renal cortex were analyzed by digital image capture followed by quantification using automated image analysis software.

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The assessment of immunohistochemical staining and apoptosis was performed as described in example 16.

Results.

Assessment of proteinuria.

To confirm the presence of proteinuria in mice, the total amount of protein excreted in the urine was analyzed in urine samples collected within 24 hours on the 15th day (end of the experiment). It was found that in urine samples, total protein levels were increased on average by almost six times in BSA-treated groups compared to non-BSA-treated control groups (data not shown), confirming the presence of proteinuria in BSA-treated mice. No significant differences were observed in protein levels between the BSA treated groups.

Assessment of histological changes.

In FIG. 32 shows typical H&E-stained tissue sections for the following groups of mice on day 15 after BSA treatment:

saline treated wild-type control mice (panel A);

control mice treated with the antibody isotype (panel B); and (panel C) wild-type mice treated with a MASP-2 inhibitory antibody (panel C).

As shown in FIG. 32, the MASP-2 inhibitory antibody treated group (panel C) showed significantly higher tissue preservation compared to the wild type treated with saline (panel A) or isotype control (panel B) at the same level of challenge. protein overdose.

Apoptosis assessment.

Apoptosis was assessed in tissue sections by staining the dUTP end-mark using terminal deoxynucleotidyl transferase (TUNEL), and the frequency of TUNEL-stained apoptotic cells was counted in sequentially selected 20 high magnification cortical fields (HPF). In FIG. 33 is a graphical representation of the frequency of TUNEL-positive apoptotic cells counted in sequentially selected 20 high magnification fields (HPFs) in renal cortical tissue sections of saline and BSA-treated control wild-type mice (n=8), wild-type mice, treated with isotype control antibody and BSA (n=8), and wild-type mice treated with MASP-2 inhibitory antibody and BSA (n=7). As shown in FIG. 33, a very significant reduction in cortical apoptosis was observed in kidneys derived from the MASP-2 inhibitory antibody group compared to the saline and isotype control group (p=0.0002 for saline vs. MASP inhibitory control). -2 antibodies; p=0.0052 for isotype control against MASP-2 inhibitory antibody).

Assessment of cytokine infiltration.

The kidney tissue sections obtained in this study evaluated interleukin 6 (IL-6), transforming growth factor beta (TGFe) and tumor necrosis factor alpha (TNFa), which are pro-inflammatory cytokines known to be activated in the proximal tubules in mice. wild-type in a model of proteinuria.

In FIG. 34 graphically shows the results of computer analysis of tissue sections stained with anti-TGFβ antibody (measured as % area of anti-TGFe antibodies stained) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIG. 34, quantification of TGFe-stained regions showed a significant reduction in TGFe levels in mice treated with inhibitory MASP-2 antibody compared to groups treated with saline and isotype control antibodies (p values=0.0324 and 0.0349, respectively).

In FIG. 35 graphically shows the results of computer analysis of images of tissue sections stained with TNFa antibody (measured as % area of anti-TNFa antibodies stained) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIG. 35, analysis of stained sections showed a significant decrease in TNFa levels in the MASP-2 inhibitory antibody group compared to the saline control group (p=0.011) as well as the isotype control group (p=0.0285).

In FIG. 36 graphically shows the results of computer analysis of images of tissue sections stained with anti-IL-6 antibody (measured as % area of anti-IL-6 stained antibodies) in wild-type mice treated with BSA and saline (n=8), mice treated with BSA and isotype control antibody (n=7), and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIG. 36, staining analysis showed a significant decrease in IL-6 levels in the MASP-2 inhibitory antibody group compared to the saline control group (p=0.0269) and the isotype control group.

- 83 043102 (p=0.0455).

General review of results and conclusions.

The results in this example demonstrate that the use of a MASP-2 inhibitory antibody provides protection against kidney damage in a protein overdose model, which is consistent with the results described in Example 16 showing that MASP-2 -/- mice have reduced kidney damage in a proteinuria model. .

Example 18.

This example presents the results that were obtained using a model of renal fibrosis, inflammation, and tubulointerstitial injury with adriamycin-induced nephrology in MASP-2 -/- mice and wild-type mice to assess the role of the lectin pathway in proteinuric nephropathy.

Background/justification.

Adriamycin is an antacycline anticancer antibiotic used to treat a wide range of cancers, including hematologic malignancies, soft tissue sarcomas, and many types of carcinomas. Adriamycin-induced nephropathy is a well-established rodent model of chronic kidney disease that has provided a better understanding of the progression of chronic proteinuria (Lee and Harris, Nephrology, 16:30-38, 2011). The pattern of structural and functional damage in adriamycin-induced nephropathy is very similar to that of chronic proteinuric kidney disease in humans (Pippin et al., American Journal of Renal Physiology, 296:F213-29, 2009).

Adriamycin-induced nephropathy is characterized by podocyte damage followed by glomerulosclerosis, tubulointerstitial inflammation, and fibrosis. Many studies have shown that adriamycin-induced nephropathy is modulated by both immune and non-immune mechanisms (Lee and Harris, Nephrology, 16:30-38, 2011). Adriamycin-induced nephropathy has several advantages as a model for kidney disease. First, it is a very reproducible and predictable model of kidney injury, as it is characterized by the induction of kidney injury within a few days of drug administration, which makes it easier to design the experiment as damage develops consistently over time. It is also a model in which the degree of tissue damage is severe despite having acceptable mortality (<5%) and morbidity (weight loss). Therefore, due to the severity and development of kidney damage over time in adriamycin-induced nephropathy, this model is suitable for testing therapies that protect against kidney damage.

As described in Examples 16 and 17, in a protein overdose-induced proteinuria model, MASP-2-/- mice and mice treated with a MASP-2 inhibitory antibody were found to have significantly improved outcomes (e.g., less tubulointerstitial damage and less inflammation). kidney), compared with wild-type mice, suggesting a pathogenic role for the lectin pathway in proteinuric kidney disease.

In this example, MASP-2 -/- mice were analyzed compared to wild-type mice in an adriamycin (AN) induced nephrology model to determine if MASP-2 deficiency can reduce and/or prevent adriamycin-induced renal inflammation and tubulointerstitial injury.

Methods.

1. Optimization of dose and time point.

First, an experiment was performed to determine the dose of adriamycin and the time point at which BALB/c mice develop a level of kidney inflammation suitable for testing a therapeutic intervention.

Three groups of wild-type BALB/c mice (n=8) were administered IV with a single dose of adriamycin (10.5 mg/kg). Mice were sacrificed at three points: one week, two weeks and four weeks after administration of adriamycin. Control mice were injected with saline only.

Results.

All mice in the three groups showed evidence of glomerulosclerosis and proteinuria as determined by H&E staining, with a gradual increase in tissue inflammation as measured by the degree of macrophage infiltration in the kidney (data not shown). The degree of tissue damage was moderate in the one week group, moderate in the two week group, and severe in the four week group (data not shown). For the remainder of the study, a two-week point was chosen.

2. Analysis of adriamycin-induced nephrology in wild-type and MASP-2 -/- mice.

To elucidate the role of the complement lectin pathway in adriamycin-induced nephrology, a group of MASP-2-/- (BALB/c) mice were compared with wild-type (BALB/c) mice at the same dose of adriamycin. MASP-2 -/- mice were crossed with BALB/c mice for 10 generations.

Wild-type mice (n=8) and MASP-2 −/− mice (n=8) were treated with IV adriamycin (10.5 mg/kg) and three mice of each strain were treated with saline alone as a control. All mice

- 84 043102 were sacrificed two weeks after treatment and tissues were collected. The extent of histopathological damage was assessed by H&E staining.

Results.

In FIG. 37 shows typical H&E-stained tissue sections for the following groups of mice on

Day 14 after treatment with adriamycin or saline alone (control):

wild-type control mice treated with saline only (panels A-1, A-2, A-3);

wild-type mice treated with adriamycin (panels B-1, B-2, B-3); And

MASP-2 -/- mice treated with adriamycin (panels C-1, C-2, C-3).

Each photo (for example, panel A-1, A-2, A-3) represents an individual mouse.

As shown in FIG. 37, in the MASP-2 -/- group treated with adriamycin, there is a much higher rate of tissue preservation compared to the wild-type group treated with the same dose of adriamycin.

In FIG. 38 graphically shows the results of computer analysis of images of kidney tissue sections stained with F4/80 macrophage marker antibody showing the mean macrophage area stained (%) for the following groups of mice on day 14 after treatment with adriamycin or saline alone (wild-type control):

wild-type control mice treated with saline only;

wild-type mice treated with adriamycin;

MASP-2 -/- mice treated only with saline; And

MASP-2 -/- mice treated with adriamycin.

As shown in FIG. 38, adriamycin-treated MASP-2 −/− mice have reduced macrophage infiltration (**p=0.007) compared to adriamycin-treated wild-type mice.

In FIG. 39 graphically shows the results of computer analysis of Sirius Red-stained kidney tissue sections showing the area of stained collagen deposition (%) for the following groups of mice on day 14 after treatment with adriamycin or saline alone (wild-type control): control wild-type mice treated with only saline; wild-type mice treated with adriamycin; MASP-2-/- mice treated with saline alone and MASP-2-/- mice treated with adriamycin. As shown in FIG. 39, MASP-2-/adriamycin-treated mice have reduced collagen deposition (**p=0.005) compared to adriamycin-treated wild-type mice.

Results and conclusions.

Relief of renal tubulointerstitial inflammation is a key goal in the treatment of kidney disease.

The results presented herein show that the lectin complement pathway contributes significantly to the development of renal tubulointerstitial inflammation. As further shown herein, a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, can be used as a novel therapeutic approach to treat proteinuric nephropathy, adriamycin-induced nephropathy, and to reduce renal tubulointerstitial inflammation.

Example 19.

This example describes the initial results of an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal inhibitory MASP-2 antibody in adults with steroid-dependent immunoglobulin-A nephropathy (IgAN) and in adults with steroid-dependent membranous nephropathy (MN).

Prerequisites.

Chronic kidney disease affects more than 20 million people in the United States (Drawz P. et al., Ann. Intern. Med., 162 (11), ITC1-16, 2015). Glomerulonephropathies (GN), including IgAN and MH, are kidney diseases in which glomeruli are damaged and often lead to end-stage renal disease and dialysis. There are several types of primary GN, the most common of which is IgAN. Many of these patients present with persistent kidney inflammation and progressive deterioration. Often these patients are treated with corticosteroids or immunosuppressants, which are associated with numerous serious long-term adverse effects. In many patients, the condition continues to worsen even with these treatments. There is no treatment for IgAN or MN.

IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is an autoimmune kidney disease that causes internal inflammation and damage to the kidneys. IgAN is the most common primary glomerulonephritis worldwide (Magistroni et al., Kidney Int., 88(5):974-89, 2015). With an annual incidence rate of approximately 2.5 per 100,000 people, it is estimated that 1 in 1,400 people in the US develop IgAN. In 40% of patients with IgAN, the disease will progress to end-stage renal disease (ESRD) within 20 years of diagnosis (Coppo R., D'Amico G., J. Nephrol.,

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18(5):503-12, 2005; Xie et al., PLoS One, 7(6):e38904 (2012)). Patients typically present with microscopic hematuria with mild to moderate proteinuria and varying levels of renal impairment (Wyatt R.J. et al., N. Engl. J. Med., 368(25):2402-14, 2013). Clinical markers such as impaired renal function, long-term hypertension, and severe proteinuria (greater than 1 g per day) are associated with poor prognosis (Goto M et al., Transplant. Nephrol. Dial., 24(10):3068-74, 2009 ; Berthoux F. et al., J. Am. Soc. Nephrol., 22(4):752-61, 2011). Proteinuria is the strongest predictor independent of other risk factors in several large nonexperimental and prospective studies (Coppo R. et al., J. Nephrol., 18(5):503-12, 2005; Reich H.N. et al. , J. Am. Soc. Nephrol., 18(12):3177-83, 2007). An estimated 15-20% of patients achieve ESRD within 10 years of onset if they are not treated (D'Amico G., Am. J. Kidney Dis., 36(2):227-37, 2000).

The diagnostic feature of IgAN is the predominance of deposition of either IgA alone or in combination with IgG, IgM, or both in the glomerular mesangium. In IgAN, kidney biopsy reveals glomerular deposition of manan-binding lectin (MBL), a key recognition molecule for activation of MASP-2, an effector enzyme of the lectin pathway of the complement system. Glomerular MBL deposits, usually co-located with IgA and indicative of complement activation, and high urinary MBL levels are associated with a poor prognosis in IgAN, with these patients showing more severe histologic changes and mesangial proliferation than those without or with high MBL deposition. urinary MBL levels (Matsuda M. et al., Nephron, 80(4):408-13, 1998; Liu L.L. et al., Clin. Exp. Immunol., 169(2):148-155, 2012; Roos A. et al., J. Am. Soc. Nephrol., 17(6):1724-34, 2006; Liu L. L. et al., Clin. Exp. Immunol., 174(1):152-60, 2013) . The remission rate is also significantly lower in patients with MBL deposition (Liu L.L. et al., Clin. Exp. Immunol., 174(1):152-60, 2013).

Current approaches to the treatment of IgAN are based on attempts to slow, stop or delay the deterioration of kidney function. The Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guideline for glomerulonephritis recommends an IgAN treatment regimen that prioritizes blood pressure control by blockade of the renin-angiotensin system (RAS) [KDIGO Work Group, 2012]. For patients with persistent daily proteinuria of 1 g or more despite maximum tolerated doses of antihypertensive agents and well controlled blood pressure, recommended treatment includes corticosteroids and/or other immunosuppressants such as cyclophosphamide, azathioprine, or mycophenolate mofetil. The Clinical Practice Guideline for Glomerulonephritis Kidney Disease: Improving Overall Outcomes (KDIGO) (Int. Soc. of Nephrol., 2(2):139-274, 2012) recommends administering corticosteroids to patients with proteinuria at a dose greater than or equal to 1 g/day with a usual duration of treatment of 6 months. For patients with sickle IgAN (defined as the presence of sickle cells in >50% of glomeruli) and rapidly deteriorating renal clearance function, another immunosuppressant (eg, cyclophosphamide) may be added to corticosteroids. However, even with aggressive immunosuppressive treatment, which is associated with severe long-term effects, some patients experience a progressive deterioration in renal function. To date, there is no FDA-approved treatment for IgAN, even when angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) are used to control blood pressure, some patients remain elevated proteinuria. None of these methods have been shown to stop or even slow the progression of IgAN in patients at risk for rapid disease progression. Alternative therapies that could reduce or eliminate the need for long-term corticosteroid and/or immunosuppressive therapy would clearly address unmet medical needs.

membranous nephropathy.

The annual incidence of membranous nephropathy (MN) is approximately 10-12 per 1,000,000 people. Patients with MR may have a variable course of the disease, but approximately 25% will develop end-stage renal disease.

Membranous nephropathy is an immunomodulated glomerular disorder and one of the most common causes of nephrotic syndrome in adults. The disease is characterized by the formation of immune deposits, primarily IgG4, on the outer layer of the glomerular basement membrane, which contain podocyte antigens and antibodies specific to these antigens, leading to complement activation. The initial manifestations of MN are associated with nephrotic syndrome: proteinuria, hypoalbuminemia, hyperlipidemia, and edema.

Although MN may improve spontaneously without treatment, one-third of patients experience progressive loss of kidney function and disease progression to ESRD at an average of 5 years after diagnosis. Corticosteroids are often used to treat MN, and there is a need to develop alternative therapies. In addition, patients who are considered at moderate risk of progression, depending on the severity of proteinuria, receive prednisone in combination with cyclophosphamide or a calcinuorin inhibitor, and these two treatments, used together, are often associated with serious systemic side effects.

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Methods.

Two phase 1 clinical studies conducted in healthy volunteers demonstrated that both intravenous and subcutaneous administration of the MASP-2 inhibitory antibody, OMS646, resulted in sustained suppression of the lectin pathway.

This example describes the interim results of an ongoing Phase 2, uncontrolled, multicentre study of a MASP-2 inhibitory antibody, OMS646, in subjects with IgAN and MH. Inclusion criteria require that all patients in this study, regardless of subtype of kidney disease, be maintained on a stable dose of corticosteroids for at least 12 weeks prior to enrollment in the study (i.e. patients were steroid dependent). The study is a non-comparative pilot study with 12 weeks of treatment and 6 weeks of follow-up.

According to the study plan, it is necessary to recruit approximately four patients per disease. The study is designed to evaluate the potential of OMS646 to improve kidney function (eg, reduce the severity of proteinuria) and reduce corticosteroid requirements in patients with IgAN and MN. To date, 2 patients with IgA nephropathy and 2 patients with membranous nephropathy have completed treatment in this study.

To enter the study, each subject must have a high level of protein in the urine, despite continuous treatment with a stable dose of corticosteroids. These criteria are chosen for patients who are unlikely to spontaneously improve during the study.

Subjects were >18 years of age at screening and subjects were only included in the study if they were diagnosed with one of the following: IgAN on kidney biopsy or primary MN on kidney biopsy. Enrolled patients also had to meet all of the following inclusion criteria (1) mean urinary albumin/creatinine ratio >0.6 in three samples collected sequentially and daily prior to each of the two visits during the selection period;

(2) taking prednisone >10 mg or equivalent dose for at least 12 weeks prior to visit 1 during the screening period;

(3) on background immunosuppressive treatment (eg, cyclophosphamide, mycophenolate mofetil), receiving a stable dose for at least 2 months prior to Visit 1 during screening with no expected dose change during the study;

(4) an estimated glomerular filtration rate (eGFR) >30 ml/min/1.73 m ² calculated from the MDRD ¹ equation;

(5) undergoing physician-prescribed, stable, optimized therapy with angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs), systolic blood pressure <150 mmHg, and diastolic blood pressure <90 mmHg. in a relaxed state;

(6) not using belimumab, eculizumab, or rituzimab within 6 months prior to visit 1 during the screening period; and (7) no kidney transplant.

1 MDRD Equation: eGFR (mL/min/1.73 ^m2 )=175χ(SCr)-1.154χ(age)-0.203χ(0.742 if female) x (1.122 if African American)

Note: SCr=Measurement of serum creatinine must be expressed in mg/dL.

The monoclonal antibody used in this study, OMS646, is a fully human IgG4 monoclonal antibody that binds and inhibits human MASP-2. MASP-2 is an effector enzyme of the lectin pathway. As shown in Example 12, OMS646 binds efficiently to recombinant MASP-2 (apparent equilibrium dissociation constant in the 100 pM range) and exhibits more than 5000-fold selectivity for homologous C1s, C1r and MASP-1 proteins. In functional assays, OMS646 inhibits the activity of the human lectin pathway with nanomole efficiency (50% inhibition concentration (IC ₅₀ ) of about 3 nM), but does not significantly affect the classical pathway. OMS646 given either by intravenous (IV) or subcutaneous (SC) injection in mice, non-human primates and humans resulted in high plasma concentrations that were associated with downregulation of lectin pathway activation in an ex vivo assay.

In this study, the drug substance OMS646 was provided at a concentration of 100 mg/ml, which was further diluted for IV administration. The corresponding calculated volume of 100 mg/mL OMS646 injection solution was withdrawn from the vial using a dosing syringe. The infusion bag was administered within 4 hours of preparation.

The study included periods of screening (28 days), treatment (12 weeks), and follow-up (6 weeks), as shown in the study design below.

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Study scheme.

During screening prior to the first dose of OMS646, screened patients provided three urine samples (which were collected once a day) in each of two three-day periods to establish baseline urinary albumin to creatinine ratios. After the screening period, selected subjects were administered IV OMS646 at 4 mg/kg once a week for 12 weeks (treatment period). The last dose of OMS646 was followed by a 6-week follow-up period.

During the first 4 weeks of treatment with OMS646, subjects continued to take the stable dose of corticosteroids they had taken prior to treatment. At the end of the first 4 weeks of a 12-week period, subjects' corticosteroid intake was tapered off (i.e., the dose of corticosteroids was reduced) if possible over a period of 4 weeks, followed by 4 weeks during which the resulting product was continued. reduced dose of corticosteroids. The aim was to reduce the dose of prednisone to <6 mg (or equivalent dose) daily. During this period, gradual dose reduction was discontinued in patients who experienced deterioration in renal function, as assessed by the investigator. Subjects were treated with OMS646 while tapering the corticosteroid dose and for a full 12 weeks. Patients were then followed up for an additional 6 weeks after the last dose of OMS646. Tapering the dose of corticosteroids and treatment with OMS646 allowed us to assess whether taking OMS646 would reduce the doses of corticosteroids needed to maintain stable renal function.

The key measures of efficacy in this study are the change in urinary albumin/creatinine ratio (uACR) and 24-hour protein levels from baseline obtained before the start of 12 weeks. Measurement of urinary protein or albumin is commonly used to evaluate kidney damage, and persistently high urinary protein levels are correlated with progression of kidney disease. uACR is used in clinical studies to assess proteinuria.

Efficiency mark.

The analyzed value of uACR is defined as the average of all values obtained at some time point. The scheduled number of uACRs is three for each scheduled time. Baseline uACR was determined as the mean of the analyzed values at two visits.

Results.

In FIG. 40 graphically shows the uACRin of two IgAN patients during a twelve week study with weekly treatment with MASP-2 inhibitory antibody (OMS646) at 4 mg/kg. As shown in FIG. 40, change from baseline is statistically significant at time point a (p=0.003), time point b (p=0.007) and time point c (p=0.033) according to untransformed analysis. In table. 12 shows protein data in a 24 hour urine collection from IgAN patients treated with OMS646.

Table 12

Protein in 24-hour urine collection (mg/day) in IgAN patients treated with OMS646

Sample time Patient 1 (mg/24 hours) Patient 2 (mg/24 hours) Average value Baseline 3876 2437 3156 Day 85 1783 455 1119 P=0.017

As shown in FIG. 40 and in table. 12, clinically and statistically significant improvement in renal function was observed in patients with IgAN during the study. Statistically significant decrease in both the uACR value (see Fig. 40) and protein concentration in a 24-hour urine collection (see Table 12). As the uACR data in FIG. 40, the mean baseline uACR was 1264 mg/g and reached 525 mg/g by the end of treatment (p=0.011), decreasing to 128 mg/g by the end of the follow-up period. In FIG. 40 additionally shows that the treatment effect was maintained throughout the observation period. Measurements of 24-hour urinary protein excretion were monitored by uACR, with a mean decrease from 3156 mg/24 h to 1119 mg/24 h (p=0.017). Treatment effects in the two patients were highly consistent.

- 88 043102

Both patients experienced a decrease of approximately 2000 mg/day, and both patients achieved partial remission (defined as greater than 50% reduction in protein excretion in 24-hour urine collection and/or final protein excretion of less than 1000 mg/day; complete remission was defined as protein excretion less than 300 mg/day). The magnitude of the 24-hour reduction in proteinuria in both patients with IgA nephropathy is associated with a significant improvement in renal survival. Both patients with IgA nephropathy were also tolerant of a substantial reduction in their steroid doses, with daily dose reductions to <5 mg (60 to 0 mg, 30 to 5 mg).

Two patients with MN also showed a decrease in uACR during treatment with OMS646. In one patient with MN, the uACR level decreased from 1003 to 69 mg/g, and this low level persisted during the follow-up period. Another patient with MN experienced a decrease in uACR from 1323 to 673 mg/g with a variable course after treatment. The first patient with MN showed a marked decrease in the protein level in the 24-hour urine collection (10771 mg/24 h at the beginning of treatment to 325 mg/24 h on day 85), which provided a partial and almost complete remission, while the other remained almost unchanged (4272 mg/24 h at the start of treatment up to 4502 mg/24 on day 85). Steroid levels in two patients with MN gradually decreased from 30 to 15 mg and from 10 to 5 mg.

Thus, consistent improvements in renal function were observed in IgAN and MH patients treated with the MASP-2 inhibitory antibody, OMS646. The effects of OMS646 treatment in IgAN patients are robust and consistent, indicating a strong signal of efficacy. These effects are confirmed by the results in patients with MN. The time profile and magnitude of uACR changes during treatment were consistent across all four IgAN and MN patients. No significant safety issues were observed. The patients in this study belonged to a difficult-to-treat group, and the therapeutic effect in these patients is indicative of the efficacy of a MASP-2 inhibitory antibody such as OMS646 in patients with IgAN and MN, such as patients suffering from steroid-dependent IgAN and MN (i.e. e. Patients treated with a stable dose of corticosteroids prior to treatment with an inhibitory MASP-2 antibody), including patients at increased risk of rapid progression to end-stage renal disease.

According to the foregoing, in one embodiment, the invention provides a method of treating a human suffering from IgAN or MN, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to suppress MASP-2 dependent complement activation. In one embodiment, the method comprises administering to a person suffering from IgAN or MH an amount of a MASP-2 inhibitory antibody sufficient to improve kidney function (eg, reduce proteinuria). In one embodiment, the subject suffers from steroid dependent IgAN. In one embodiment, the subject suffers from steroid-dependent MN. In one embodiment, the MASP-2 inhibitory antibody is administered to a subject suffering from steroid-dependent IgAN or steroid-dependent MN in an amount sufficient to improve renal function and/or reduce the dose of corticosteroids in said subject.

In one embodiment, the method further comprises identifying a subject suffering from steroid dependent IgAN prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to suppress MASP-2 dependent complement activation.

In one embodiment, the method further comprises identifying a subject having steroid dependent MN prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to suppress MASP-2 dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a K _D of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2 , said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ of 10 nM or less, said antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ of 3 0 nM 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 , where the antibody is a single chain molecule, where said antibody is an IgG2 molecule, where said antibody is an IgGl molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without essentially inhibiting the classical pathway (ie, 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 kidney function, such as reduction in proteinuria (e.g., reduction in uACR and/or reduction in 24-hour protein concentration). urine collection, such as greater than 20% reduction in protein excretion in a 24-hour urine collection, or such as greater than 30% reduction in protein excretion in a 24-hour urine collection, or such as greater than 40% reduction in protein excretion in a 24-hour urine collection collection of urine or, such as

- 89 043102 more than 50% reduction in protein excretion in a 24-hour urine collection).

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from IgAN (such as steroid-dependent IgAN) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to a week or two weeks, or three weeks, or four weeks or more), followed by subcutaneous administration of the MASP-2 inhibitory antibody to the subject for a second period of time (eg, chronic phase for at least two weeks or more).

In some embodiments, the method includes administering a MASP-2 inhibitory agent to a subject suffering from MN (such as steroid-dependent MN) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to a week or two weeks, or three weeks or four weeks or more) followed by subcutaneous administration of the MASP-2 inhibitory antibody to the subject for a second period of time (eg, chronic phase for at least two weeks or more).

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from IgAN (such as steroid-dependent IgAN) or MN (such as steroid-dependent MN) intravenously, intramuscularly, or subcutaneously. The treatment may be chronic and administered daily monthly, but preferably at least every two weeks or at least once a week, for example twice a week or three times a week.

In one embodiment, the method comprises treating a subject suffering from IgAN (such as steroid dependent IgAN) or MN (such as steroid dependent MN) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen binding fragment containing a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67 and a light chain variable region containing CDR-Ll, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69 In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (I) a) a heavy chain variable region comprising

i) a heavy chain CDR-H1 containing the amino acid sequence from aa 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from aa 50-65 in SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from aa 95-107 in SEQ ID NO: 67; And

b) a light chain variable region containing

i) a light chain CDR-L1 containing the amino acid sequence from aa 24-34 in SEQ ID NO: 69; and ii) a light chain CDR-L2 comprising the amino acid sequence from aa 50-56 in SEQ ID NO: 69; and iii) a light chain CDR-L3 comprising the amino acid sequence from aa 89-97 in SEQ ID NO: 69; or (II) a variant thereof comprising a heavy chain variable region with at least 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) with SEQ ID NO: 67, and a light chain variable region with at least 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 least 99% identity) with SEQ ID NO: 69.

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

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

In some embodiments, the method includes administering to a subject suffering from or at risk of developing IgAN (such as steroid dependent IgAN) or MN (such as steroid dependent MN) a composition comprising a MASP-2 inhibitory antibody or an antigen binding fragment containing a heavy chain variable region containing an amino acid the sequence shown in SEQ ID NO: 67 and a light chain variable region containing an amino acid

- 90 043102 lot sequence shown in SEQ ID NO: 69, at a dose of 1 to 10 mg/kg (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg ) at least once a week (for example, at least twice a week or at least three times a week) for at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks , or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks, or for at least 9 weeks, or for at least 10 weeks, or for at least 11 weeks, or for at least 12 weeks.

Example 20.

This example describes the preliminary results of a phase 2 ongoing clinical study evaluating the safety and clinical efficacy of a fully human monoclonal inhibitory MASP-2 antibody in adults with steroid dependent lupus nephritis (LN).

Prerequisites.

More than 20 million people in the United States suffer from chronic kidney disease (Drawz P. et al., Ann. Intern. Med., 162 (11), ITC1-16, 2015). Glomerulonephropathy (GN), including IgAN, MH, and BH, are kidney diseases in which glomeruli are damaged, often leading to end-stage renal disease and dialysis. Many of these patients have persistent kidney inflammation and progressive deterioration. Often these patients are treated with corticosteroids or immunosuppressants, which are associated with numerous long-term serious adverse effects. Even with these treatments, many patients continue to deteriorate.

Lupus nephritis.

The main complication of systemic lupus erythematosus (SLE) is nephritis, also known as lupus nephritis, which is classified as a secondary form of glomerulonephritis. Later in the course of the disease, up to 60% of adults with SLE develop some form of kidney damage (KodaKimble et al., Koda-Kimble and Young's Applied Therapeutics: the clinical use of drugs, 10th ed., Lippincott Williams & Wilkins, p. 792 -9, 2012) with a frequency of 20-70 per 100,000 people in the USA. Lupus nephritis is often accompanied by other symptoms of active SLE in patients, including fatigue, fever, rash, arthritis, serositis, or central nervous system disease (Pisetsky D.S. et al., Med. Clin. North. Am., 81(1):113-28 , 1997). Some patients have asymptomatic lupus nephritis; however, according to routine monitoring, laboratory abnormalities such as elevated serum creatinine, low albumin, or protein or urinary sediment are indicative of active lupus nephritis. Autoimmunity plays an important role in the pathogenesis of lupus nephritis. These autoantibodies form intravascular pathogenic immune complexes that are deposited in the glomeruli. Autoantibodies can also bind to antigens already located in the glomerular basement membrane, forming immune complexes in situ. Immune complexes promote the inflammatory response by activating complement and recruiting inflammatory cells (D'Agati V.D. et al., Lupus nephritis: pathology and pathogenesis: Wallace D.J. Hahn, Dubois' Lupus Erythematosus, 7th ed., Philadelpha: Lippincott Williams & Wiklins, p. 1094-111, 2007). Thus, immune complex-mediated complement activation plays a key role in the pathogenesis of lupus nephritis. C4d deposits are present in renal tissue and are usually associated with immune complex deposits, C1q and C3, initiating the classical pathway. In some cases, C4d deposits are present without C1q, indicating possible involvement of the lectin pathway (Kim M.K. et al., Int. J. Clin. Exp. Pathol., 6(10):2157-67, 2013).

An additional confirmation of the important role of the lectin pathway is the fact that MBL deposits occur in the lesions of the skin of patients with SLE (Wallim L.R. et al., Hum. Immunol., 75(7):629-32, 2014). In addition, persistent deposition of MBL and ficolins was observed in most kidney biopsies from patients with lupus nephritis (Nisihara R.M. et al., Hum. Immunol., 74(8):907-10, 2013). MBL deposition in the kidneys was most pronounced in patients with high proteinuria. In addition, plasma MBL levels were significantly higher in SLE patients than healthy controls, and MBL levels correlated with disease activity, suggesting that MBL levels may represent a biomarker of SLE disease activity (Panda A.K. et al., Arthritis Res. Ther., 14(5):R218, 2012). Corticosteroids are the main traditional treatment option for patients with mild lupus nephritis. In more severe cases, high doses of prednisone, methylprednisolone, mycophenolate mofetil, cyclophosphamide, azathioprine, and cyclosporine have been used in clinical practice. Treatment options for SLE and lupus nephritis are associated with high morbidity and mortality. Side effects, especially with long-term use of corticosteroids, limit patient compliance with the treatment regimen with a subsequent impact on its effectiveness. There is a need to develop treatment regimens that are more easily tolerated by patients.

Methods.

As described in Example 19 above, two phase 1 clinical studies conducted in healthy volunteers demonstrated that both intravenous and subcutaneous administration of an inhibitory

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The MASP-2 antibody, OMS646, resulted in sustained downregulation of the lectin pathway.

This example describes the interim results from a phase 2 ongoing, uncontrolled, multicentre study of a MASP-2 inhibitory antibody, OMS646, in patients with lupus nephritis (LN). In accordance with the inclusion criteria for the study, all patients in this study, regardless of the subtype of kidney disease, must receive a stable dose of corticosteroids for at least 12 weeks prior to their inclusion in the study (i.e. patients are steroid-dependent). The study is a simple (no comparison) pilot study with a 12-week course of treatment and a 6-week follow-up period.

The study is designed to evaluate the ability of OMS646 to improve kidney function (eg, reduce proteinuria) and reduce the need for corticosteroids in patients with LN. To date, 5 patients with lupus nephritis (LN) have completed treatment in the study.

At the start of the study, each subject should have a high level of protein in the urine despite ongoing treatment with a stable dose of corticosteroids. These criteria select patients who are unlikely to spontaneously improve during the study period.

Patients were >18 years of age at screening, and only those patients diagnosed with lupus nephritis on renal biopsy were included in the study. Enrolled patients also had to meet all of the following inclusion criteria (1) mean urine albumin/creatinine ratio >0.6 in three samples taken consecutively and daily prior to each of the 2 visits during the screening period;

(2) taking >10 mg prednisone or an equivalent dose for at least 12 weeks prior to Screening 1;

(3) in the case of immunosuppressive therapy (eg, cyclophosphamide, mycophenolate mofetil), taking a stable dose for at least 2 months prior to Screening Visit 1 with no expected dose change during the study;

(4) an estimated glomerular filtration rate (eGFR) >30 ml/min/1.73 m ² calculated from the MDRD ¹ equation;

(5) receiving, under the guidance of a physician, stable, optimized treatment with angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs), and systolic blood pressure <150 mmHg. and diastolic blood pressure <90 mmHg. at rest;

(6) no use of belimumab, eculizumab, or rituzimab within 6 months of Screening Visit 1; and (7) no history of kidney transplantation.

¹ MDRD Equation: eGFR (mL/min/1.73m ² )=175χ(SCr) ¹ . ¹⁵⁴ χ(Age) ⁰ . ²⁰³ χ (0.742 (for women)) x (1.212 (for African Americans))

Note: SCr=serum creatinine should be expressed in mg/dl.

The monoclonal antibody used in this study, OMS646, is a fully human IgG4 monoclonal antibody that binds to and inhibits human MASP-2. MASP-2 is an effector enzyme of the lectin pathway. As shown in Example 12, OMS646 binds strongly to recombinant MASP-2 (apparent equilibrium dissociation constant of 100 pM) and exhibits more than 5000-fold selectivity for homologous C1s, C1r and MASP-1 proteins. In functional assays, OMS646 inhibits the human lectin pathway with nanomolar activity (the concentration resulting in 50% inhibition [IC50] is approximately 3 nM), but does not significantly affect the classical pathway. In an ex vivo assay, high plasma concentrations of OMS646 administered by intravenous (IV) or subcutaneous (SC) injection to mice, non-human primates and humans resulted in downregulation of lectin pathway activation.

In this study, the drug substance OMS646 was provided at a concentration of 100 mg/ml, which was then diluted for intravenous administration. The corresponding calculated volume of the 100 mg/mL OMS646 injection solution was withdrawn from the vial with a dosing syringe. The infusion bag was administered within 4 hours of preparation.

The study consists of screening (28 days), treatment (12 weeks), and follow-up (6 weeks) periods as shown in the study outline below.

Informed Consent

Treatment

Observation

I i ' ' ' ' ' % ί...................Dosing w i Dosing | I Weeks M

Screening Weeks 1-4 | Reduced visit 1 and 2 .----Maintenance , _{+ d} „ _a ^Nedep > steroids . i steroids I up to 6 mg or , . , , _e ,. , .. , ., , . - & equivalent | Dosing

I Weeks 9-12 ΐ Maintenance -¼ reduced | t doses of steroids

Post-treatment visit Week 13

Ϊ Follow-up visit

P observation | Week 16

Visit at the end of the study

Week 18

During the screening period and prior to the administration of the first dose of OMS646, subjects who met the inclusion criteria in the study provided three urine samples (collected once a day) on three consecutive days in each of two urine collection periods for three consecutive days to determine baseline protein in a 24-hour urine collection; and urinary albumin-creatinine ratio. After the screening period, eligible subjects received OMS646 at 4 mg/kg intravenously once a week for 12 weeks (treatment period). The last dose of OMS646 was followed by a 6-week follow-up period.

During the first 4 weeks of treatment with OMS646, subjects received a stable pre-study dose of corticosteroids. At the end of the first 4 weeks of the 12-week treatment period, subjects received a tapered dose of corticosteroids (i.e., the dose of corticosteroids was reduced), if possible, for 4 weeks, followed by 4 weeks, during which subjects continued to receive the final dose of corticosteroids. The aim was to reduce the daily dose of prednisone to 6 mg (or equivalent dose). During this period, the dose reduction was stopped for those subjects who, in the opinion of the investigator, there was a deterioration in renal function. Subjects received OMS646 treatment at the time of corticosteroid dose reduction and for all 12 weeks of the treatment period. Patients were then followed up for an additional 6 weeks after the last dose. Dose reduction of corticosteroids and treatment with OMS646 allowed us to evaluate the ability of OMS646 to reduce the dose of corticosteroids required to maintain stable renal function.

Performance analysis.

The key measure of effectiveness in this study is the change in 24-hour protein levels from baseline over 12 weeks. Measurement of urinary protein or albumin is commonly used to assess kidney damage, and persistently high urinary protein levels are correlated with progression of kidney disease. Partial remission is defined as a greater than 50% reduction in 24-hour urinary protein excretion.

Results.

In table. 13 shows the protein level in a 24-hour urine collection (mg/day) for five LN patients treated with OMS646.

Table 13

Protein level in 24-hour urine collection (mg/day) in patients with LN treated with OMS646

Sample time Patient #1 (mg/ 24h) Patient No. 2 (mg/ 24h) Patient #3 (mg/24 h) Patient #4 (mg/24h) Patient #5 (mg/24h) Average value (patients #2-5) Baseline 1112 7539.0 2066.9 2217.8 4067.0 15890.7 Day 85 13731 2750.9 282.0 1168.0 797.6 4998.5

Note. Patient #1 experienced a sudden exacerbation of a systemic disease during treatment.

As shown in Table. 13, patients with LN showed a clinically and statistically significant improvement in renal function throughout the study. As shown in Table. 13, four of five patients with LN experienced a significant (median 69%) reduction in 24-hour urinary protein excretion during treatment. The fifth patient (Patient #1) experienced a sudden exacerbation of a systemic disease with a marked increase. For the majority of respondents with lupus, their doses of steroids were reduced.

Thus, a significant improvement in kidney function was observed in four out of five LN patients treated with the MASP-2 inhibitory antibody, OMS646. The results of treatment with OMS646 in patients with LN were consistent and significant, indicating a strong signal of efficacy. No significant safety issues were observed. In this study, patients represent a difficult-to-treat group, and it is suggested that the therapeutic effect observed in these patients is a predictor of the efficacy of a MASP-2 inhibitory antibody, such as OMS646, in patients with LN, such as patients suffering from steroid-dependent LN (i.e. e. Patients treated with a stable dose of corticosteroids prior to initiation of treatment with an inhibitory MASP-2 antibody), including patients at risk for rapid progression of kidney disease to end stage.

Accordingly, in one embodiment, the invention provides a method of treating a human subject suffering from LN, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to suppress MASP-2 dependent complement activation. In one embodiment, the method comprises administering to a human subject suffering from LN an amount of a MASP-2 inhibitory antibody sufficient to improve kidney function (eg, to reduce proteinuria). In one embodiment, the subject is suffering from steroid-dependent LN. In one embodiment, the MASP-2 inhibitory antibody is administered to a subject suffering from steroid-dependent LN in an amount sufficient to improve the subject's kidney function and/or reduce the dose of corticosteroids.

In one embodiment, the method further comprises identifying a human subject suffering from steroid-dependent LL prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to suppress MASP-2-dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a K _D of 10 nM or less, said antibody binds an epitope in the CCP1 domain MASP-2, said antibody inhibits C3b deposition in an in vitro assay of 1% human serum at an _IC50 of 10 nM or less, said antibody inhibits C3b deposition in 90% human serum at an _IC50 of 30 nM or less, wherein the antibody is a fragment an antibody 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 said antibody is an IgG2 molecule, wherein said antibody is an IgG1 molecule , and the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (ie, inhibits the lectin pathway while leaving the classical complement pathway intact).

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject suffering from LN in an amount effective to improve at least one or more clinical parameters associated with kidney function, such as the reduction of proteinuria (for example, a decrease in uACR and/or a decrease in the concentration protein in a 24-hour urine collection, such as a decrease in 24-hour urinary protein excretion by more than 20% or a decrease in 24-hour urinary protein excretion by more than 30%, or a decrease in 24-hour urinary protein excretion by more than 40 %, or, for example, a decrease in 24-hour urinary protein excretion by more than 50%). In some embodiments, a MASP-2 inhibitory antibody is administered to a subject suffering from LN in an amount effective to produce at least partial remission of proteinuria (i.e., greater than a 50% reduction in 24-hour urinary protein excretion compared to baseline).

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from LN (such as a steroid-dependent LN) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day per week, two weeks, three weeks or four weeks or more), followed by subcutaneous administration of the MASP-2 inhibitory antibody for a second period of time (eg, a chronic phase lasting at least two weeks or more).

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from LN (such as a steroid-dependent LN), intravenously, intramuscularly, or subcutaneously. The treatment may be long-term and may be daily or monthly, but preferably at least every two weeks or at least once a week, for example twice a week or three times a week.

In one embodiment, the method comprises treating a subject suffering from LN (such as a steroid-dependent LN) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof containing a heavy chain variable region comprising CDR-H1, CDR- H2 and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69. In some embodiments, the composition contains an inhibitory MASP-2 antibody containing (I) (a) a heavy chain variable region containing

i) a heavy chain CDR-H1 containing the amino acid sequence of 31-35 of SEQ ID NO: 67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95-107 of SEQ ID NO: 67; And

b) a light chain variable region containing

i) a light chain CDR-L1 containing the amino acid sequence of 24-34 of SEQ ID NO: 69;

ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO: 69; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO: 69; or (II) a variant thereof comprising a heavy chain variable region with at least 90% identity

- 94 043102 accuracy (for example, 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) with SEQ ID NO: 67; and a light chain variable region with at least 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) with SEQ ID NO: 69.

In some embodiments, the method comprises administering to a subject suffering from LN (such as a steroid-dependent LN) a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67 , and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69.

In some embodiments, the method comprises administering to a subject suffering from LN (such as a steroid-dependent LN) a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by reference antibody OMS646 containing the heavy chain variable region as set forth in SEQ ID NO: 67 and the light chain variable region as set forth in SEQ ID NO: 69.

In some embodiments, the method comprises administering to a subject suffering from or at risk of developing LN (such as steroid-dependent LN) a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. : 67, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69 at a dose of 1 to 10 mg/kg (i.e. 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 mg/kg) at least once a week (for example, at least twice a week or at least three times a week) for a period of 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.

Example 21.

This example describes additional results from an ongoing phase 2 clinical trial evaluating the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody, OMS646, in reducing proteinuria in adult patients with glomerulopathies, including IgAN, as described in example 19.

Methods.

As described in Example 19, Phase 2 involved IgAN patients receiving corticosteroids at the time of their enrollment in the study; all patients received open-label OMS646 and two patients with IgAN were positive. Currently, two more IgAN patients have completed the dosing step as described in Example 19, and a total of four IgAN patients have completed the study.

To be included in this study, patients with IgAN had to have (1) biopsy-diagnosed IgAN;

(2) uACR >0.6 g/g;

(3) eGFR >30 ml/min/1.73 m ² ;

(4) controlled blood pressure during treatment with a stable dose of ACEI/ARB; and (5) taking a stable dose of the steroid prednisone >10 mg for at least 12 weeks.

All four adult IgAN patients treated with OMS646 in this study had previously diagnosed renal failure with an estimated glomerular filtration rate (eGFR) of 30 to 46 ml/mg/1.73 m ² and a 24-hour protein level of 2.44 up to 4.87 g/24 hours. By the beginning of the study, all patients received stable blockade of the renin-angiotensin system (RAS) and at least 3 months of treatment with corticosteroids.

All patients received OMS646 IV once a week for 12 weeks. Patients underwent a four-week run-in period followed by treatment with OMS646, which included four weeks of stable steroid dose, four weeks of steroid dose reduction, if possible, and four weeks of reduced steroid dose. After treatment with OMS646, patients were followed up for an additional 6 weeks of the study. After the completion of the study, the investigator continued to follow up the patients.

The following parameters were measured to evaluate efficacy in the study:

(1) urinary albumin/creatinine ratio (uACR) measured 6 times before treatment (baseline) and 3 times at each efficacy evaluation during treatment and follow-up; and (2) protein level in 24-hour urine collection measured once before treatment with OMS646 and once

- 95 043102 2-4 weeks after completion of OMS646 treatment.

According to the protocol, the dose of corticosteroids was reduced between 4 and 8 weeks, if this was clinically appropriate.

Results.

Four IgAN patients completed a 6-week post-treatment follow-up period. In table. 14 presents the demographic and baseline characteristics of these patients.

Table 14

Demographics and background characteristics

Patient 1 Patient 2 Patient 3 Patient 4 Age (years) 35 32 55 45 Floor Male female female female Race White Asian White White Time since diagnosis 8 years 5 months 5 months 2 years uACR (mg/g) 1271 1256 1674 1628 24 hours protein level (mg) 3876 2437 4872 4554 eGFR (ml/min/SSA) 46 thirty 44 42 Arterial 110/84 124/74 126/74 136/66 pressure (mmHg. )

eGFR - estimated glomerular filtration rate;

SSA standard surface area (1.73 ^m2 );

uACR - urine albumin/creatinine ratio.

All patients showed a marked reduction in proteinuria during treatment with OMS646. Statistically and clinically significant improvements were seen in both uACR and 24-hour protein levels, as shown in FIG. 41 and 42.

In FIG. 41 is a plot of uACR (mg/g) versus time for four IgAN patients treated with OMS646 from baseline to day 120. As shown in FIG. 41, from baseline to end of study, mean uACR decreased by 1.13 g/g±0.27 (77% reduction, p=0.026). As further shown in FIG. 41, at the last visit during follow-up after treatment with OMS646, each patient's uACR decreased by 94%, 86%, 47%, and 89% (patients 1-4, respectively) from baseline.

In FIG. 42 graphically shows the change in protein level in the post-treatment 24-hour urine collection from baseline on pre-treatment day 1 in four IgAN patients treated with OMS646. As shown in FIG. 42, the protein level in the 24 hour urine collection decreased by 54%, 81%, 63%, and 95% (patients 1-4, respectively) from baseline.

In FIG. 43 graphically shows the mean change in protein level in the 24 hour post-treatment urine collection from baseline pre-treatment levels for four IgAN patients treated with OMS646. As shown in FIG. 43, the average protein level in the 24-hour urine collection decreased by 2.87±1.08 g/day (73% reduction; p=0.013).

All patients were able to stop taking corticosteroids during or shortly after the study, indicating that the effect of OMS646 on proteinuria is unlikely to be due to corticosteroids. Estimated glomerular filtration rates (eGFRs) (calculated from the Renal Disease Diet Modification Formula) were stable throughout the treatment period and the follow-up period.

OMS646 was well tolerated by all patients.

Thus, in phase 2 of this open-label clinical study, a significant and sustained decrease in uACR was observed in all IgAN patients treated with OMS646 for 12 weeks. 24-hour proteinuria was significantly reduced in all patients. The observed degree of reduction in proteinuria has been associated with significant improvements in kidney disease prognosis and clinical outcomes (Inker L.A. et al., Am. J. Kidney. Dis., 68(3):392-401 (2016)). The results demonstrating a strong reduction in proteinuria allowing steroid withdrawal after treatment with OMS646, a monoclonal anti-MASP-2 antibody that inhibits the action of the complement lectin pathway, support the use of OMS646 as a therapeutic agent to improve

- 96 043102 results of treatment of IgA glomerulopathy. The effects of OMS646 in IgAN patients are consistent and significant, suggesting its efficacy in this population.

Example 22.

Maintenance of remission after completion of OMS646 treatment in patients with IgA nephropathy (IgAN).

Prerequisites.

According to examples 19 and 21, in the phase 2 IgAN patient studies, four IgAN patients were treated with OMS646, a fully human monoclonal antibody that inhibits MASP-2 activity. As described in examples 19 and 21, all patients received OMS646 IV once a week for 12 weeks. According to example 21, after treatment with OMS646, study patients were followed up for an additional 6 weeks, and all IgAN patients treated with OMS646 experienced partial remission (defined as a decrease of more than 50% in 24-hour urinary protein excretion and/or final protein excretion less than 1000 mg/day). As described in this example, after completion of the study, these four patients were followed up to assess the duration of remission after treatment with OMS646.

Methods.

After completion of the phase 2 clinical studies described in example 21, the researcher observed the status of 4 patients with IgAN treated with OMS646. The study endpoints were uACR and 24-hour proteinuria. As described in Example 21, all four IgAN patients were in partial remission at the end of the study. At follow-up, urinary protein to creatinine ratio (uPCR) was measured. Each uPCR value was converted to uACR (urinary albumin/creatinine ratio) by multiplying by 0.64 (see Zhao et al., Clin. J. Am. Soc. Nephrol., 11:947-55, 2016).

Results.

All patients showed partial remission after treatment with OMS646. The average age of three women and one man was 42; three of them were Europeans and one was Asian. The mean eGFR was 41 ml/min/1.73 m ² and the mean steroid dose administered was 55 mg. The follow-up period after the last dose of OMS646 was 2 to 10 months. As described in Example 21, the mean uACR decreased by 77% during the study (p=0.026). During follow-up, three patients remained in partial remission (54%, 93%, and 78% reduction in uACR at 12, 12, and 5 months, respectively). In one patient, the uACR value was 88% of baseline at 7 months. During follow-up, three patients also experienced eGFR improvements of 7, 13, and 7 ml/min/1.73 m ² . eGFR in the fourth patient was stable. All patients stopped taking steroids. OMS646 was well tolerated.

Thus, as described in Example 21, proteinuria was significantly reduced in IgAN patients during the 12-week OMS646 treatment and the 6-week post-treatment follow-up period included in the study. This decrease in proteinuria was maintained up to 10 months after completion of treatment. These data support the use of OMS646 as a therapeutic agent to improve the outcome of IgA glomerulopathy.

An update from the investigator on the status of the four patients described in this example at approximately one year of follow-up after a single 12-week course of treatment with OMS646 reported that three of the four patients maintained a reduction in proteinuria. In these three patients, uACR remained reduced by 14%, 23%, and 24% from baseline, which were in patients before OMS646 treatment. In addition, an improvement in estimated glomerular filtration rate (eGFR), which is a measure of kidney function, was seen in 3 out of 4 patients after the study. In the patient with the most pronounced decrease in renal function, an improvement in eGFR from 30 ml/min/1.73 m ² to 47 ml/min/1.73 m ² was observed, i.e. the improvement was 57%.

Thus, the sustained reduction in proteinuria after completion of one course of OMS646 treatment remained impressive during one year of follow-up. The improvement observed in the eGFR value was unexpected, especially after a year of follow-up, as it was expected that this would take a longer period of time. As described above, two of the four patients showed a slight increase in eGFR, and the response in one of the patients was an impressive 50% improvement. The improvements observed in eGFR indicate that an additional beneficial effect of OMS646 on patients is the potential elimination or significant delay of the need for dialysis, as well as a reduction in the risk of complications associated with the progression of chronic kidney disease.

In accordance with the foregoing, in one embodiment, the invention relates to a method for reducing proteinuria in a human subject suffering from IgAN, comprising administering to the subject a MASP-2 inhibitory antibody or antigen-binding fragment containing a heavy chain variable region containing CDR-H1, CDR -H2 and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69, according to the following dosing schemes:

- 97 043102

c) administering about 4 mg/kg (ie, 3.6 to 4.4 mg/kg) of said antibody to an IgAN-suffering subject intravenously once a week for a treatment period of at least weeks; or

d) administering about 180 mg to about 725 mg (i.e., 162 to 797 mg) of said antibody to an IgAN-suffering subject intravenously once a week for a treatment period of at least 12 weeks, wherein the method reduces proteinuria in specified human subject.

In one embodiment, the dose of the MASP-2 inhibitory antibody is about 4 mg/kg (i.e., 3.6 to 4.4 mg/kg), such as about 3.6 mg/kg, about 3.7 mg/kg. kg, about 3.8 mg/kg, about 3.9 m/kg, about 4.0 mg/kg, about 4.1 mg/kg, about 4.2 mg/kg, about 4.3 mg/kg, or about 4.4 mg/kg.

In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of about 180 mg to about 725 mg (i.e., 160 to 800 mg or about 300 to 500 mg, e.g., about 300 mg to about 400 mg ), e.g., about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 315 mg, about 320 mg, about 325 mg, about 330 mg, about 335 mg, about 340 mg about 345 mg, about 350 mg About 355 mg About 360 mg About 365 mg About 370 mg About 375 mg About 380 mg About 385 mg About 390 mg About 395 mg About 400 mg About 405 mg About 410 mg , about 415 mg, about 420 mg, about 425 mg, about 430 mg, about 435 mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460 mg, about 465 mg, about 470 mg, about 475 mg, about 480 mg, about 485 mg, about 490 mg, about 495 mg, about 500 mg about 505 mg, about 510 mg, about 515 mg, about 520 mg, about 525 mg, about 530 mg, about 535 mg, about 540 mg, about 545 mg, about 550 mg, about 555 mg, about 560 mg, about 565 mg, about 570 mg, about 575 mg, about 580 mg, about 585 mg, about 590 mg, about 595 mg, about 600 mg, about 605 mg, about 610 mg, about 615 mg, about 620 mg, about 625 mg, about 630 mg, about 635 mg, about 640 mg, about 645 mg, about 650 mg, about 655 mg, about 660 mg, about 665 mg, about 670 mg, about 675 mg, about 680 mg, about 685 mg, about 690 mg, about 695 mg, about 700 mg, about 705 mg, about 710 mg, about 715 mg, about 720 mg, about 725 mg, about 730 mg, about 735 mg, about 740 mg, about 745 mg, about 750 mg about 755 mg, about 760 mg, about 765 mg, about 770 mg, about 775 mg, about 780 mg, about 785 mg, about 790 mg, about 795 mg, or about 800 mg.

In one embodiment, the treatment period is 12 weeks.

In one embodiment, the treatment period is followed by a break (i.e., a period without administration of a MASP-2 inhibitor) of at least 2 months, or a break of at least 3 months, or a break of at least 4 months , or a break of at least 5 months, or a break of at least 6 months or longer, such as a break of at least 7 months, or a break of at least 8 months, or a break of at least at least 9 months, or a break of at least 10 months, or a break of at least 11 months, or a break of at least 12 months or longer.

In some embodiments, the method further comprises periodically monitoring the subject's urinary protein levels during the treatment period and/or the break, and optionally resuming treatment with the MASP-2 inhibitory antibody upon detection of a recurrence of proteinuria.

In some embodiments, the method is effective to reduce proteinuria in a subject suffering from IgAN by at least 30%, such as at least 40% or at least 50% or more than 50% of baseline (before treatment) , according to the results at the end of the treatment period and/or at the end of the break.

In some embodiments, the method is effective to increase the estimated glomerular filtration rate (eGFR) in a subject suffering from IgAN.

In some embodiments, the subject suffering from IgAN has proteinuria greater than 1 g protein/24 hour urinary protein excretion prior to treatment, and the method is effective to reduce the subject's proteinuria by at least 30%, e.g., at least 40%, or at least 50% or more than 50% of baseline (pre-treatment) as measured at the end of the treatment period and/or at the end of the break, and/or to reduce proteinuria to less than 1 g of protein/24- hourly urinary protein excretion, according to the results at the end of the treatment period and / or at the end of the break.

- 98 043102

In some embodiments, the subject suffering from IgAN has a proteinuria greater than 1 g protein/24-hour urinary protein excretion despite maximum tolerated doses of antihypertensive agents and well-controlled blood pressure prior to treatment, and the method is effective in reducing the subject's proteinuria by at least 30%, such as at least 40%, or at least 50%, or more than 50% of the baseline (before treatment) determined at the end of the treatment period and/or at the end of the break and/or to reduce proteinuria to less than 1 g of protein / 24-hour urinary protein excretion, determined at the end of the treatment period and / or at the end of the break.

In some embodiments, the subject suffering from IgAN has not been treated with steroids for at least one year. In some embodiments, the subject suffering from IgAN receives steroid treatment for at least a portion of the 12 week OMS646 treatment period. In some embodiments, the subject suffering from IgAN receives steroid treatment for at least a portion of the 12 week OMS646 treatment period and the method is effective in reducing proteinuria and reducing or eliminating the need for steroid treatment by the end of the treatment period and/or by the end of the break.

Other embodiments.

All publications, patent applications and patents mentioned in this description are incorporated into this description by reference.

Various modifications and variations of the described 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. While the invention has been described with reference to specific preferred embodiments, it should be understood that the claimed invention should not be limited in any way to such specific embodiments.

In accordance with the foregoing, the invention provides the following embodiments.

1A. A method for treating, suppressing, alleviating, or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused by or complicated by fibrosis and/or inflammation, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to suppress fibrosis.

2A. The method according to paragraph 1A, wherein the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof.

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

4A. The method according to paragraph 2A, wherein the MASP-2 antibody or fragment thereof specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity 10 times greater than that of another antigen in the complement system.

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

6A. The method of paragraph 1A, wherein the MASP-2 inhibitory agent selectively inhibits complement lectin pathway activation without significantly inhibiting C1q-dependent complement activation.

7A. The method according to paragraph 1A, wherein the MASP-2 inhibitory agent is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, or by inhalation.

8A. The method according to any one of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is associated with ischemic reperfusion injury.

9A. The method according to any one of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is not associated with ischemic reperfusion injury.

10A. The method according to any one of paragraphs 1A-7A, wherein prior to the administration of the MASP-2 inhibitory agent, the subject was suffering from proteinuria and the administration of the MASP-2 inhibitory agent reduces the subject's proteinuria.

11A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the kidneys.

12A. The method according to paragraph 11A, wherein the MASP-2 inhibitory agent is administered in an amount effective to suppress tubulointerstitial fibrosis.

13A. The method of paragraph 11A, wherein the MASP-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in the subject.

14A. The method according to paragraph 11A, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, chronic renal failure, glomerular disease (e.g. focal segmental glomerulosclerosis), immune complex

- 99 043102 devices (eg IgA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial lesions and glomerulonephritis (eg C3 glomerulopathy).

15A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the lungs.

16A. The method according to paragraph 15A, wherein the disease or disorder is selected from the group consisting of chronic obstructive pulmonary disease, cystic fibrosis, scleroderma-associated pulmonary fibrosis, bronchiectasis, and pulmonary hypertension.

17A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the liver.

18A. The method according to paragraph 17A, wherein the disease or disorder is selected from the group consisting of cirrhosis, non-alcoholic fatty liver disease (steatohepatitis), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to acute or chronic hepatitis, biliary disease, and liver toxicity (eg, drug-induced hepatotoxicity caused by acetaminophen or another drug such as nephrotoxin).

19A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the heart.

20A. The method of paragraph 19A, wherein the disease or disorder is selected from the group consisting of cardiac fibrosis, myocardial infarction, valvular fibrosis, atrial fibrosis, endomyocardial fibrosis, arrhythmic right ventricular cardiomyopathy (ARCH).

21A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by vascular fibrosis.

22A. The method according to paragraph 21A, wherein the disease or disorder is selected from the group consisting of vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis, and abdominal aortic aneurysm.

23A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by skin fibrosis.

24A. The method according to paragraph 23A, wherein the disease or disorder is selected from the group consisting of excessive wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarring, and hypertrophic scars.

25A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by joint fibrosis.

26A. The method according to paragraph 25A, wherein the disease or disorder is arthrofibrosis.

27A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of the central nervous system.

28. The method of paragraph 27A, wherein the disease or disorder is selected from the group consisting of stroke, traumatic brain injury, and spinal cord injury.

29A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of the digestive system.

30A. The method of paragraph 29A, wherein the disease or disorder is selected from the group consisting of Crohn's disease, pancreatic fibrosis, and ulcerative colitis.

31A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by ocular fibrosis.

32A. The method according to paragraph 31A, wherein the disease or disorder is selected from the group consisting of anterior subcapsular cataract, posterior capsular opacity, macular degeneration, and retinal and vitreal retinopathy.

33A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of a bone or soft tissue structure of the musculoskeletal system.

34A. The method according to paragraph 33A, wherein the disease or disorder is selected from the group consisting of osteoporosis and/or osteopenia associated with cystic fibrosis, myelodysplastic conditions with increased bone fibrosis, cellular capsulitis, Dupuytren's contracture, and myelofibrosis.

35A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of the reproductive organs.

36A. The method according to paragraph 35A, wherein the disease or disorder is selected from the group consisting of endometriosis and Peyronie's disease.

37A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a chronic infectious disease caused or complicated by fibrosis and/or inflammation.

38A. The method according to paragraph 37A, wherein the infectious disease is selected from the group consisting of alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza.

39A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from an autoimmune disease caused or complicated by fibrosis and/or inflammation.

40A. The method according to paragraph 39A, wherein the autoimmune disease is selected from the group consisting of scleroderma and systemic lupus erythematosus (SLE).

41A. The method according to any one of paragraphs 1A-7A, wherein the subject is suffering from trauma-related scarring.

42A. The method according to paragraph 41A, wherein the trauma-related scarring is selected from the group consisting of surgical complications (e.g., surgical adhesions in which scar tissue may form between internal organs, causing contracture, pain, and may lead to infertility) induced by chemotherapy means of fibrosis, radiation-induced fibrosis and scarring associated with burns.

43A. The method according to any one of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of organ transplantation, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis , bladder fibrosis and pleural fibrosis.

1b. A method for preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, comprising administering an amount of a MASP-2 inhibitory agent effective to reduce or prevent proteinuria in the subject.

2b. The method according to paragraph 1B, wherein the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or fragment thereof.

3b. The method according to paragraph 1B or 2B, wherein the MASP-2 inhibitory agent is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion compared to baseline 24-hour protein excretion in the urine of the subject prior to treatment.

4b. The method according to any one of paragraphs 1B-3B, wherein the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, exlampsia, kidney toxicity, amyloidosis, vascular collagen diseases (e.g., systemic lupus erythematosus), lupus nephritis, dehydration, glomerular disease (eg, membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy (eg , Berger's disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport's syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold, and other heavy metals, ACE inhibitors, antibiotics (eg adriamycin) or opiates (eg heroin) or other nephrotoxins); Fabry disease, infections (eg, HIV, syphilis, hepatitis A, B, or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), ebola hemorrhagic fever, nail patella syndrome, familial fever, HELLP syndrome, systemic lupus erythematosus, granulomatosis Wegener, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, Henoch-Schoenlein purpura, urinary tract infection that has spread to the kidneys, Sjögren's syndrome, and post-infectious glomerulonephritis.

5b. The method according to any one of paragraphs 1B-3B, wherein the disease or condition associated with proteinuria is IgA nephropathy (ie, Berger's disease).

6b. The method according to any one of paragraphs 1B-3B, wherein the disease or condition associated with proteinuria is membranous nephropathy.

7B. The method according to any one of paragraphs 1B-3B, wherein the disease or condition associated with proteinuria is lupus nephritis.

1C. A method for inhibiting the progression of chronic kidney disease, comprising administering to a subject in need thereof an amount of a MASP-2 inhibitory agent effective to reduce or prevent tubulointerstitial fibrosis of the kidney.

2C. The method according to paragraph 1C, wherein the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or fragment thereof.

3C. The method according to paragraph 1C, wherein the subject in need has proteinuria prior to administration of the MASP-2 inhibitory agent and administration of the MASP-2 inhibitory agent reduces proteinuria in the subject such that the subject has at least a 20% reduction in 24 hour protein excretion with urine compared with the initial 24-hour excretion of protein in the urine of the specified subject prior to treatment.

4C. The method according to paragraph 1C, wherein the MASP-2 inhibitory agent is administered in an amount effective

- 101 043102 effective for reducing, delaying or eliminating the need for dialysis in a subject.

1D. A method for protecting the kidneys from kidney damage in a subject who has received, is receiving or will receive treatment with one or more nephrotoxic agents, comprising administering an amount of a MASP-2 inhibitory agent effective to prevent or alleviate drug-induced nephropathy.

2D. The method according to paragraph 1D, wherein the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or fragment thereof.

3D. The method according to paragraph 1C, wherein the MASP-2 inhibitory agent is administered prior to administration of said nephrotoxic agent.

4D. The method according to paragraph 1C, wherein the MASP-2 inhibitory agent is administered together with said nephrotoxic agent.

5D. The method according to paragraph 1C, wherein the MASP-2 inhibitory agent is administered after said nephrotoxic agent to treat nephrotoxicity.

1E. A method of treating a human suffering from immunoglobin A nephropathy (IgAN), comprising administering to a subject a composition containing an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to suppress MASP-2-dependent complement activation.

2E. The method according to paragraph 1E, where the subject suffers from steroid-dependent IgAN.

3E. The method according to paragraph 1E or 2E, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2.

4E. The method according to any one of paragraphs 1E-3E, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

5E. The method according to any one of paragraphs 1E-4E, wherein the MASP-2 inhibitory antibody does not substantially suppress the classical pathway.

6E. The method according to any one of paragraphs 1E-5E, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC _{50 of} 30 nM or less.

7E. The method according to paragraph 2E, further comprising identifying a person having a steroid-dependent IgAN, prior to administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve kidney function.

8E. The method according to any one of paragraphs 1E-7E, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount effective to improve kidney function.

9E. The method of paragraph 8E, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion from baseline 24- hourly urinary protein excretion in the subject prior to treatment.

10E. The method according to paragraph 1E, where the composition is administered in an amount sufficient to improve kidney function and reduce the dose of corticosteroids in the specified subject.

11E. The method according to any one of paragraphs 1E-10E, 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 shown in SEQ ID NO: 67, and the variable a light chain region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69.

1F. A method of treating a human suffering from membranous nephropathy (MN), comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to suppress MASP-2-dependent complement activation.

2F. The method according to paragraph 1F, wherein the subject suffers from steroid-dependent MN.

3F. The method according to paragraph 1F or 2F, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2.

4F. The method according to any of paragraphs 1F-3F, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

5F. The method according to any one of paragraphs 1F-4F, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

6F. The method according to any one of paragraphs 1F-5F, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC _{50 of} 30 nM or less.

7F. The method of paragraph 1F, wherein the method further comprises identifying a human subject having steroid-dependent MN prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve kidney function.

8F. The method according to any one of paragraphs 1F-7F, wherein the MASP-2 inhibitory antibody or an-

- 102 043102 tigen-binding fragment is administered in an amount effective to improve kidney function.

9F. The method of paragraph 8F, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion from baseline 24- hourly urinary protein excretion in the subject prior to treatment.

10F. The method according to paragraph 1F or 2F, wherein the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in said subject.

11F. The method according to any one of paragraphs 1F-10F, 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 shown in SEQ ID NO: 67, and the variable a light chain region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69.

1g. A method of treating a human subject suffering from lupus nephritis (LN), comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, effective to suppress MASP-2-dependent complement activation.

2g. The method according to paragraph 1G, wherein the subject suffers from steroid-dependent LN.

3g. The method according to paragraph 1G or 2G, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2.

4G. The method according to any one of paragraphs 1G-3G, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

5G. The method according to any one of paragraphs 1G-4G, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

6g. The method according to any one of paragraphs 1G-5G, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC _{50 of} 30 nM or less.

7g. The method of paragraph 1G, wherein the method further comprises identifying a human subject having a steroid-dependent LL prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve kidney function.

8g. The method according to any one of paragraphs 1G-7G, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount effective to improve kidney function.

9g. The method of paragraph 8G, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion from baseline 24- hourly urinary protein excretion in the subject prior to treatment.

10g. The method according to paragraph 1G or 2G, wherein the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in said subject.

11g. The method according to any one of paragraphs 1G-10G, 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 shown in SEQ ID NO: 67, and the variable a light chain region containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 69.

While illustrative embodiments have been shown and described, it will be understood that various changes may be made without departing from the spirit and scope of the invention.

Claims (6)

1. A method of treating a subject suffering from steroid-dependent lupus nephritis (LN), comprising administering to the subject a composition comprising a MASP-2 inhibitory monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to human MASP-2 and suppresses MASP-2-dependent complement activation in an amount effective to improve kidney function, wherein said 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. 2. The method of claim 1, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. 3. The method of claim 1, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ of 3 0 nM or less. 4. The method of claim 1, wherein the method further comprises identifying a human subject having a steroid-dependent VL prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve - 103 043102 kidney function. 5. The method of claim 1, wherein the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount and for a period of time that is effective to achieve at least a 20% reduction in 24-hour urinary protein excretion compared to with the subject's baseline 24-hour urinary protein excretion prior to treatment. 6. The method of claim 1, wherein the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in said subject. and/d-EGF-like