CN118530341A

CN118530341A - Method for improving the effectiveness of a drug comprising an immunoglobulin Fc fragment

Info

Publication number: CN118530341A
Application number: CN202410609791.1A
Authority: CN
Inventors: 张鹏; 李百勇; 夏瑜; 王忠民
Original assignee: Akeso Biopharma Inc
Current assignee: Akeso Pharmaceuticals Inc
Priority date: 2021-03-12
Filing date: 2022-03-11
Publication date: 2024-08-23
Also published as: CN115073588B; CN115073588A; WO2022188867A1; CN118530342A

Abstract

The invention belongs to the field of tumor treatment and molecular immunology, and relates to a method for improving the effectiveness of a medicine containing an immunoglobulin Fc fragment. The invention also relates to a method of reducing or blocking the level of IL-8 and/or IL-6 secreted by immune cells mediated by a drug comprising an Fc fragment of an immunoglobulin. The invention can effectively improve the safety and/or effectiveness of medicaments containing immunoglobulin Fc fragments.

Description

Method for improving the effectiveness of a drug comprising an immunoglobulin Fc fragment

The present invention is a divisional application of the parent application with the application number 202210243178.3, the application date of the parent application is 2022, 3 and 11, and the invention name is "a method for improving the safety of a drug containing an immunoglobulin Fc fragment".

Technical Field

The invention belongs to the field of tumor treatment and molecular immunology, and relates to a method for optimizing a drug (such as an antibody or Fc fusion protein) containing an immunoglobulin Fc fragment and improving the safety and/or effectiveness of the drug. In particular, the invention relates to a method of reducing or blocking the level of IL-8 and/or IL-6 secreted by immune cells mediated by a drug (e.g., an antibody or Fc fusion protein) containing an Fc fragment of an immunoglobulin.

Background

Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells or somatic cells for the recognition of immune responses mediated by the Fc region of antibodies. After the antibody Fab region recognizes an antigen, its antibody Fc region binds to an Fc receptor on an immune cell (e.g., a killer cell) to activate an effector cell.

Depending on the type of antibody recognized by the Fc receptor and the cell expressing it, fc receptors are largely classified into three types fcγ R, fc αr and fcεr, which in turn can be classified into four subtypes fcγri (also known as CD 64), fcγrii (also known as CD 32), fcγriii (also known as CD 16) and FcRn (also known as Neonatal Fc receptor). Wherein fcγri, fcγrii, fcγriii are closely related to ADCC effect. Fcγriii is the predominant molecule mediating ADCC, with two subtypes of fcγriiia and fcγriiib being highly homologous in different cell types, and the high affinity fcγriiia subtype caused by the single nucleotide stem polymorphism (SNP) site is present in the fcγriiia population, referred to as fcγriiia_v158 and the low affinity fcγriiia_f158 subtype, respectively. Fcγri has higher affinity to the Fc region of IgG, involved in ADCC process; fcγrii has three subtypes fcγriia, fcγriib and fcγriic (also referred to as CD32a, CD32b, CD32c, respectively), wherein fcγriia has ADCC activity; fcyriia exists in the population in two subtypes due to single nucleotide mutations, called fcyriia_h131 and fcyriia_r131, respectively (Hogarth PM, pietersga.2012, nature Review Drug Discovery,11 (4): 311-331).

The IgG family comprises four members, igG1, igG2, igG3 and IgG4, whose crystallizable fragment (fragment crystallizable, fc) regions of the heavy chain constant region differ in amino acid, resulting in their affinities for fcγrs being different. IgG1 is the most abundant subtype in humans, and is also the most abundant subtype used in monoclonal antibodies, igG1 being able to bind various fcγrs. IgG2 has the weakest affinity for fcγrs, but IgG2 is still able to bind to fcγriia. IgG3 binds most strongly to fcγrs. IgG4 molecules bind poorly to fcγrs other than fcγri. IgG4 antibody subtypes are unstable and are susceptible to cleavage of hinge regions leading to Fab-arm exchange, forming half-molecules and bispecific, functionally monovalent antibodies (Aalberse R.C.et al Clin. Exp. Allergy.2009;39 (4): 469-77.); introduction of an S228P mutation in the heavy chain hinge region of an IgG4 antibody stabilizes the IgG4 molecule, preventing the formation of half-molecules (Shirley JPeters et al.J Biol chem.2012; 287 (29): 24525-33.).

ADCR (anti-DEPENDENT CYTOKINE RELEASE) refers to antibody-dependent cytokine release, the Fab fragment of an antibody binds to an epitope of a tumor cell, its Fc fragment cross-binds to an effector cell surface Fc Receptor (FcR), and activation of the effector cell by crosslinking results in massive secretion of cytokines such as IL (interleukin) -1, IL-6, IL-8, IL-10, MCP (monocyte chemotactic protein) -1 etc. release, wherein IL-6 is the main inflammatory mediator. These cytokines can impair the efficacy of immunotherapy while increasing immune-related adverse reactions, which can severely lead to multiple organ failure and death.

Interleukin 8 (interleutin-8, IL-8) is a chemotactic cytokine (Chemotactic cytokines) belonging to the CXC-alpha subfamily (also known as CXCL-8). In normal human body, it is secreted by mononuclear cells, immune cells, epithelial cells, etc., and participates in inflammation and immune defensive reaction in vivo; the receptor (CXCR) is a dimeric glycoprotein consisting of two subunits of 59 and 67kDa, belonging to the superfamily of G protein-coupled receptors, of which there are two subtypes, CXCR1 and CXCR2, respectively. IL-8 plays an important role in the proliferation of normal cells and tumor cells, and in particular has an important promoting effect on the occurrence and development of tumors. Studies have shown that IL-8 can promote the development of tumors; tumor cells themselves also secrete IL-8, promoting tumor growth and metastasis (Lo MC et al cancer letters,2013,335 (1): 81-92.). Therefore, IL-8 has become an important inflammatory factor that is indispensable in the tumor microenvironment.

IL-8 as a pro-inflammatory factor is closely related to the development and progression of tumors. In the malignant transformation process of the non-kidney cancer cells induced by the methyl arsonic acid (methylarsonate), the expression of the IL-8 gene is increased, the IL-8 gene silencing can obviously inhibit the growth of transplanted tumors in mice, and in addition, the reduction of the IL-8 level can inhibit the expression of matrix metalloproteinase-9 (Matrix metalloproteinase-9), cyclin D1 (Cyclin D1), pro-apoptotic protein Bcl-2 and Vascular Endothelial Growth Factor (VEGF) related to the growth and metastasis of the tumors (Escudero-Lourdes C et al.toxicology AND APPLIED pharmacology,2012,258 (1): 10-18). Inoue et al have found that IL-8 induces malignant transformation and invasive increases in non-neoplastic bladder cell lines (233 JP) and that there is a significant decrease in the probability of malignant transformation of 233JP cells in IL-8 knockout mice (Inoue K et al cancer Res,2000,60 (8): 2290-2299). Furthermore, in prostate cancer, IL-8 can promote the occurrence of castration-resistant prostate cancer (CRPC) in patients (Chen K et al cancer research,2015,75 (10): 1992-2004), and is associated with resistance to tumor therapy (Araki S et al cancer Res,2007,67 (14): 6854-6862.); gene silencing of IL-8 or its receptor can induce tumor cell cycle arrest, inhibiting proliferation of tumors (Singh RK, lokeshwar BL. Molecular Cancer,2009, 8:57.). The above studies indicate that IL-8 levels are closely related to the development and progression of tumors. Further studies (Mian BM et al Clin CANCER RES,2003,9 (8): 3167-3175.) indicate that IL-8 can be a new target for tumor therapy. In a tumor model of bladder cancer, the use of anti-IL-8 antibodies can significantly inhibit tumor growth.

IL-6 is produced rapidly by macrophages, responds to pathogen-associated molecular patterns (PAMP) or injury-associated molecular patterns (DAMP), and cures damaged tissue by removing infectious agents, inducing an acute phase and immune response, and is protective.

IL-6 is also responsive to compounds that include Toll-like receptor (TLR) ligands and pro-inflammatory cytokines such as IL-1 and TNF- α. In infectious lesions, IL-6 is produced by stimulation of TLRs on monocytes and macrophages, each of which recognizes a corresponding bacterial, viral or fungal component such as lipopolysaccharide, cpG DNA, double or single stranded RNA and peptidoglycan, PAMP. IL-6 can also be produced in non-infectious inflammatory conditions such as burns and wounds, the level of which depends on the severity of the disease. Damaged or necrotic cells and damaged or degraded extracellular matrix are released by the DAMP pattern, such as mitochondrial DNA, high mobility group box chromosomal protein (HMGB 1), heat shock proteins, and S100 molecules, etc., and then stimulate the corresponding TLRs to produce pro-inflammatory cytokines including IL-6.

Although IL-6 plays an important role in the resistance and repair of infection and tissue damage, high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells, thereby inhibiting myocardial function, and can even cause "cytokine storms" that produce severe acute systemic inflammatory responses. Cytokine storm is a fatal complication and adverse reaction in viral infection, tumor immunotherapy, and the like.

Immune-related adverse effects are a common and dangerous adverse effect in anti-tumor therapy with immune checkpoint inhibitors (immune checkpoint inhibitor, ICI) (Spain Let al.cancer Treat rev.2016; 44:51-60.). In recent years, immune checkpoint inhibitors have achieved great success in tumor immunotherapy, but have also resulted in a completely new toxicity profile due to off-target effects. Data in which serious immune-related adverse events (irAE), especially of major organs including heart, lung and brain, may be life threatening (Bergqvist V,et al.Cancer Immunol Immunother.2017;66(5):581-592.;Gomatou G et al.Respiration.2020;1:1-11.;Joshi MN et al.Clin Endocrinol(Oxf).2016;85(3):331-9.;Prieux-Klotz C et al.Target Oncol.2017;12(3):301-308.;Tajiri K et al.Jpn J Clin Oncol.2018;48(1):7-12.). have shown that ICI may induce off-target effects through 4 mechanisms, including direct binding to normal cell surface expressed immune checkpoint molecules, activating complement hypersensitivity; normal tissues and tumor cells have homologous antigens/epitopes; generating autoantibodies; increasing the levels of pro-inflammatory cytokines, such as IL-6 et al (MARTINS F ET al, THE LANCET Oncology,20 (1), e54-e 64).

Current anti-IL-6 therapies, such as tolizumab, a recombinant humanized anti-IL-6R monoclonal antibody, have been used to treat acute stage severe irAE, severe or refractory arthritis, macrovasculitis, uveitis, myocarditis, pneumonia, myasthenia gravis, etc. (MARTINS F ET al., THE LANCET Oncology,20 (1), e54-e 64).

Fc fusion protein drugs, such as IL-2-Fc fusion proteins, have been demonstrated to be useful in the treatment of tumors, however, due to their inherent greater toxic effects, such as IL-2-Fc fusion proteins can induce lethal capillary leakage and induce proliferation of immunosuppressive Treg cells, affecting their anti-tumor activity, and if their Fc fragments can further induce immune cells to secrete IL-8 and/or IL-6, they will significantly affect their anti-tumor effectiveness and safety. These all limit their clinical application.

In summary, aiming at immune checkpoint inhibitors and Fc fusion protein drugs, particularly antibody drugs targeting immune checkpoints and Fc fusion protein drugs taking cytokines, chemokines and ligands thereof as action mechanisms, inhibiting the effect of inducing immune cells to secrete IL-8 and/or IL-6 has great significance for improving the effectiveness and/or safety of the drugs.

Disclosure of Invention

Through intensive research and creative labor, the inventor carries out corresponding modification on the Fc end of an anti-double immune checkpoint inhibitor (anti-PD-1/CTLA 4 bispecific antibody, anti-PD-1/CD 73 bispecific antibody, anti-PD-1/LAG 3 bispecific antibody and the like, or an Fc fusion protein with immunoregulatory biological activity, such as a fusion protein of IL-2 and Fc), and can effectively reduce or eliminate the activity of IL-6 and/or IL-8 secreted by unintended immune cells mediated or induced by an immune checkpoint therapeutic antibody or fusion protein, thereby increasing the safety and/or effectiveness of the immune checkpoint inhibitor and the fusion protein drug. The following invention is thus provided:

One aspect of the invention relates to a method for reducing the level of IL-8 and/or IL-6 secreted by immune cells mediated or induced by a drug comprising an Fc fragment of an immunoglobulin, comprising the steps of:

According to the EU numbering system, the immunoglobulin Fc fragment comprises the following mutations:

L234A and L235A;

L234A and G237A;

L235A and G237A;

Or alternatively

L234A, L a and G237A.

In some embodiments of the invention, the method, wherein the drug comprising an immunoglobulin Fc fragment comprises an antibody and/or an Fc fusion protein;

optionally, the immunoglobulin Fc fragment-containing medicament further comprises one or more pharmaceutically acceptable excipients.

In some embodiments of the invention, the method wherein the drug comprising an immunoglobulin Fc fragment is an antibody.

In some embodiments of the invention, the method wherein the drug comprising an immunoglobulin Fc fragment is an Fc fusion protein.

In some embodiments of the invention, the method wherein the drug comprising an immunoglobulin Fc fragment is an antibody or Fc fusion protein.

In some embodiments of the invention, the method, wherein the drug comprising an immunoglobulin Fc fragment comprises an antibody and/or Fc fusion protein as an active ingredient (API), and one or more pharmaceutically acceptable excipients.

In some embodiments of the invention, the method wherein the drug containing an immunoglobulin Fc fragment consists of an antibody and/or Fc fusion protein as an active ingredient (API), and one or more pharmaceutically acceptable excipients.

In some embodiments of the invention, the method wherein the drug containing an immunoglobulin Fc fragment comprises an antibody and/or Fc fusion protein as the sole active ingredient (API), and one or more pharmaceutically acceptable excipients.

In some embodiments of the invention, the method wherein the drug containing an immunoglobulin Fc fragment consists of an antibody and/or Fc fusion protein as the sole active ingredient (API), and one or more pharmaceutically acceptable excipients.

Those skilled in the art will appreciate that when the immunoglobulin Fc fragment-containing medicament contains one or more pharmaceutically acceptable excipients, it is actually a pharmaceutical composition. Can be prepared into various dosage forms, such as injection, etc., according to the skills of the person skilled in the art.

In some embodiments of the invention, the method wherein the antibody is an immune checkpoint inhibitor.

In some embodiments of the invention, the method wherein the antibody is a bispecific antibody or a multispecific antibody.

In some embodiments of the invention, the method, wherein the antibody targets:

PD-1 and CTLA4, PD-1 and CD73, PD-1 and LAG3, CTLA4 and CD73, CTLA4 and LAG3, or CD73 and LAG3.

In some embodiments of the invention, the method, wherein the bispecific antibody targets PD-1 and CTLA4, comprising:

A first protein functional region targeting PD-1, and

A second protein functional region that targets CTLA 4;

Wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin;

Wherein,

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively; and the heavy chain variable region of the single chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 38-40 respectively, and the light chain variable region of the single chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 41-43 respectively;

Or alternatively

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 38-40 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 41-43 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively;

Wherein,

The immunoglobulin is human IgG;

the number of the single-chain antibodies is two, and one end of each single-chain antibody is respectively connected with the C ends of two heavy chains of the immunoglobulin.

In some embodiments of the invention, the method, wherein,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 30; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 16, SEQ ID NO. 20 and SEQ ID NO. 31;

Or alternatively

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 30; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 16, SEQ ID NO. 20 and SEQ ID NO. 31; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64.

In some embodiments of the invention, the method, wherein the bispecific antibody is selected from any one of (1) - (18) below:

(1)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 4; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 16;

(2)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 4; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 20;

(3)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 4; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 31;

(4)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 8; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 16;

(5)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 8; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 20;

(6)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 8; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 31;

(7)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 16;

(8)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 20;

(9)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 31;

(10)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 4;

(11)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 8;

(12)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 16; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 64;

(13)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 4;

(14)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 8;

(15)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 20; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 64;

(16)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 31; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 4;

(17)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 31; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 8;

(18)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 31; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 64.

In some embodiments of the invention, the method, wherein the bispecific antibody targets CD73 and PD-1, comprises:

A first protein functional region targeting CD73, and

A second protein functional region targeting PD-1;

Wherein,

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 44-46 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 47-49 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively;

Or alternatively

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 44-46 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 47-49 respectively;

Wherein,

The immunoglobulin is human IgG;

In some embodiments of the invention, the method, wherein,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 22; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 26; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64;

Or alternatively

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64; and the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO. 22; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO. 26.

In some embodiments of the invention, the method, wherein the bispecific antibody is selected from any one of (1) - (6) below:

(1)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 26; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 4;

(2)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 26; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 8;

(3)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 26; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 64;

(4)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 4; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 26;

(5)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 8; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 26;

(6)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 26.

In some embodiments of the invention, the method, wherein the bispecific antibody targets LAG3 and PD-1, comprising:

a first protein functional region targeting LAG3, and

A second protein functional region targeting PD-1;

Wherein,

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 50-52 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 53-55 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively;

Or alternatively

The heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 50-52 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 53-55 respectively;

Wherein,

The immunoglobulin is human IgG;

In some embodiments of the invention, the method, wherein,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 57; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO. 59; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO.4, SEQ ID NO. 8 and SEQ ID NO. 64;

Or alternatively

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 2 and SEQ ID NO. 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO.4, SEQ ID NO. 8 and SEQ ID NO. 64; and the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO. 57; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO. 59.

(1)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 59; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 4;

(2)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 59; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 8;

(3)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 59; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO. 64;

(4)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 4; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 59;

(5)

The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 8; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 59;

(6)

the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown as SEQ ID NO. 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region of the single chain antibody is shown as SEQ ID NO. 59.

In some embodiments of the invention, the method wherein the heavy chain constant region of the immunoglobulin of the bispecific antibody is selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 and the light chain constant region of the immunoglobulin of the bispecific antibody is selected from the light chain constant region of human IgG1, igG2, igG3 or IgG 4;

Preferably, the heavy chain constant region of the immunoglobulin of the bispecific antibody is a human Ig gamma-1chain C region or a human Ig gamma-4chain C region, and the light chain constant region of the immunoglobulin of the bispecific antibody is a human IG KAPPA CHAIN C region.

When the antibody employs the heavy chain constant region and the light chain constant region described above, the immunoglobulin Fc fragment of the bispecific antibody comprises the aforementioned mutation, i.e., the heavy chain constant region of the immunoglobulin of the bispecific antibody comprises the aforementioned mutation. For example:

In some embodiments of the invention, the method wherein the heavy chain constant region of the immunoglobulin of the bispecific antibody is selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 and the light chain constant region of the immunoglobulin of the bispecific antibody is selected from the light chain constant region of human IgG1, igG2, igG3 or IgG 4; and the heavy chain constant region of the immunoglobulin of the bispecific antibody comprises the following mutations according to the EU numbering system:

L234A and L235A;

L234A and G237A;

L235A and G237A;

Or alternatively

L234A, L a and G237A.

Also for example:

In some embodiments of the invention, the method, wherein the heavy chain constant region of the immunoglobulin of the bispecific antibody is human Ig gamma-1chain C region or human Ig gamma-4chain C region, and the light chain constant region of the immunoglobulin of the bispecific antibody is human IG KAPPA CHAIN C region; and the heavy chain constant region of the immunoglobulin of the bispecific antibody comprises the following mutations according to the EU numbering system:

L234A and L235A;

L234A and G237A;

L235A and G237A;

Or alternatively

L234A, L a and G237A.

In some embodiments of the invention, the method, wherein the immune cell is a human immune cell, e.g., a human macrophage.

In some embodiments of the invention, the method, wherein the method is a method of non-therapeutic purpose.

In some embodiments of the invention, the method, wherein the method is a method for pharmaceutical purposes.

In some embodiments of the invention, the method is a pharmaceutical method.

Another aspect of the invention relates to a method of increasing the effectiveness and/or safety of a drug comprising an immunoglobulin Fc fragment, wherein the level of IL-8 and/or IL-6 secreted by a drug-mediated or induced immune cell comprising an immunoglobulin Fc fragment is reduced by the method of any one of the invention. In some embodiments of the invention, the method is a pharmaceutical method.

It is known to those skilled in the art that the variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs) (the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3, and the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which are named by Kabat et al, see Sequences of Proteins of Immunological Interest, fifth Edition (1991), volume 1-3, NIH Publication 91-3242, bethesda Md).

The amino acid sequences of the CDR regions of the antibody sequences according to the invention are analyzed by means known to the person skilled in the art, for example by the VBASE2 database, with the following results:

(1)14C12

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

The amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

HCDR1:GFAFSSYD(SEQ ID NO:32)

HCDR2:ISGGGRYT(SEQ ID NO:33)

HCDR3:ANRYGEAWFAY(SEQ ID NO:34)

The amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:

LCDR1:QDINTY(SEQ ID NO:35)

LCDR2:RAN(SEQ ID NO:36)

LCDR3:LQYDEFPLT(SEQ ID NO:37)

(2)14C12H1L1(hG1WT)

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

The amino acid sequence of the 3 CDR regions of the heavy chain variable region is identical to 14C 12.

The amino acid sequence of the 3 CDR regions of the light chain variable region is identical to 14C 12.

(3)4G10

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16;

HCDR1:GYSFTGYT(SEQ ID NO:38)

HCDR2:INPYNNIT(SEQ ID NO:39)

HCDR3:ARLDYRSY(SEQ ID NO:40)

LCDR1:TGAVTTSNF(SEQ ID NO:41)

LCDR2:GTN(SEQ ID NO:42)

LCDR3:ALWYSNHWV(SEQ ID NO:43)

(4)4G10H3L3

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20;

The amino acid sequence of the 3 CDR regions of the heavy chain variable region is identical to that of 4G 10.

The amino acid sequence of the 3 CDR regions of the light chain variable region is identical to that of 4G 10.

(5)4G10H3V(M)L3V(M)

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 31;

(6)19F3H2(hG1TM)

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 26;

HCDR1：GYSFTGYT(SEQ ID NO:44)

HCDR2：INPYNAGT(SEQ ID NO:45)

HCDR3：ARSEYRYGGDYFDY(SEQ ID NO:46)

LCDR1：QSLLNSSNQKNY(SEQ ID NO:47)

LCDR2：FAS(SEQ ID NO:48)

LCDR3：QQHYDTPYT(SEQ ID NO:49)

(7)H7L8

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 57, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 59;

HCDR1：GGSISDYY(SEQ ID NO:50)

HCDR2：INHRGTT(SEQ ID NO:51)

HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:52)

LCDR1：QTISSY(SEQ ID NO:53)

LCDR2：DAS(SEQ ID NO:54)

LCDR3：QQRSNWPIT(SEQ ID NO:55)

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, when referring to the amino acid sequence of the PD-1 protein (Programmed CELL DEATH protein 1), it includes, but is not limited to, the full length of the PD-1 protein (NCBI GenBank: np_ 005009.2), or the extracellular fragment PD-1ECD of PD-1 or a fragment comprising PD-1 ECD; also included are fusion proteins of PD-1 ECDs, such as fragments fused to Fc protein fragments of mouse or human IgG (mFc or hFc). However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of the PD-1 protein without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" shall include all such sequences, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of a PD-1 protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.

As used herein, when referring to the amino acid sequence of CTLA4 protein, it includes, but is not limited to, the full length of CTLA4 protein (NCBI Genebank ID: np_ 054862.1), or the extracellular fragment CTLA4 ECD of CTLA4 or a fragment comprising CTLA4 ECD; also included are fusion proteins of CTLA4 ECD, e.g., fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally occurring or artificially introduced in the amino acid sequence of CTLA4 protein without affecting its biological function. Thus, in the present invention, the term "CTLA4 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing sequence fragments of CTLA4 proteins, including CTLA4 sequence fragments, also include corresponding sequence fragments in natural or artificial variants thereof.

As used herein, when referring to the amino acid sequence of the CD73 protein, it includes, but is not limited to, the full length of the CD73 protein (NCBI Genebank ID: np_ 054862.1), or the extracellular fragment of CD73 ECD or a fragment comprising CD73 ECD; also included are fusion proteins of CD73 ECD, e.g., fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it will be appreciated by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of the CD73 protein without affecting its biological function. Thus, in the present invention, the term "CD73 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of a CD73 protein, it includes a fragment of the CD73 sequence, as well as corresponding fragments of the sequence in natural or artificial variants thereof.

As used herein, when referring to the amino acid sequence of LAG3 protein, it includes, but is not limited to, the full length of LAG3 protein (NCBI Genebank ID: np_ 002277.4), or the extracellular fragment LAG3 ECD of LAG3 or a fragment comprising LAG3 ECD; also included are fusion proteins of LAG3 ECD, e.g., fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of LAG3 protein without affecting its biological function. Thus, in the present invention, the term "LAG3 protein" shall include all such sequences, as well as natural or artificial variants thereof. And, when describing sequence fragments of LAG3 proteins, including LAG3 sequence fragments, also include corresponding sequence fragments in natural or artificial variants thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable using the hybridoma technique first reported by Kohler et al (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, e.g., U.S. patent 4,816,567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (recipient antibody) with those of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some of the amino acid residues of the Framework Regions (FR) of the recipient antibody may also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies see, e.g., ,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); and Clark, immunol. Today 21:397 402 (2000).

As used herein, the term "isolated" or "isolated" refers to obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" or "separated" does not exclude the presence of substances mixed with artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substances.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with an affinity (KD) of less than about 10 ^-5 M, such as less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less.

As used herein, the term "K _D" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Typically, the antibody binds to an antigen (e.g., PD-1 protein) with a dissociation equilibrium constant (K _D) of less than about 10 ^-5 M, e.g., less than about 10 ^-6M、10^-7M、10^-8M、10^- ⁹ M or 10 ^-10 M or less. K _D can be determined using methods known to those skilled in the art, for example using a Fortebio molecular interaction instrument.

As used herein, the terms "monoclonal antibody" and "mab" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, e.g., pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, e.g., tween-80, ionic strength enhancers include, but are not limited to sodium chloride.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (e.g., a tumor); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "complete elimination" refers to detection by existing instrumentation (e.g., fortebio oct molecular interactor) that shows no binding signal or very low binding signal. In one embodiment of the invention, a binding signal that is very low means that the binding signal is below 0.1nm.

In the present invention, the term "Fusion Protein (FP)" means a protein product obtained by purposefully ligating two or more genes encoding functional proteins together to express a desired protein, by ligating the coding regions of two or more genes end to end under artificial conditions, and expressing the genes constituted by the same regulatory sequences.

In the present invention, the term "immunoglobulin (Ig) fusion protein" refers to a recombinant protein having the above two-part domains, which is expressed in eukaryotic or prokaryotic cells by linking a gene of interest to an Ig-part fragment gene at the gene level. Proteins of interest can be divided into two major classes, depending on their linkage to different fragments of Ig: one class is Fab (Fv) fusion proteins; the other class is Fc fusion proteins.

In the present invention, the term "Fc fusion protein" refers to a novel protein produced by fusing a functional protein molecule having biological activity, which may be a soluble ligand (or receptor) molecule capable of binding an endogenous receptor (or ligand) or other active substance (e.g., cytokine) requiring an extended half-life, to an Fc fragment using genetic engineering or the like. The Fc fusion protein mainly combines bioactive proteins with the hinge region and CH2 and CH3 regions of Ig.

The term "Fc fragment" or "Fc fragment", also known as a crystallizable fragment (fragment crystallizable).

In the present invention, the term "cytokine" is a small molecular weight regulatory protein secreted by a cell that affects the behavior (activation, proliferation, differentiation, migration, etc.) of the cell.

In the present invention, the term "chemokine (chemokines)" is a special class of cytokines, low molecular weight proteins that affect leukocyte chemotaxis and other cellular behavior, and plays an important role in the middle of inflammatory responses.

In the present invention, letters before a site denote amino acids before mutation, and letters after a site denote amino acids after mutation, unless otherwise specified.

Advantageous effects of the invention

The present invention achieves one or more of the technical effects described in the following items (1) to (2):

(1) The invention can effectively inhibit, reduce or eliminate the secretion of IL-6 and/or IL-8 of immune cells mediated or induced by antibody medicines;

(2) The present invention can also exert an effect of effectively eliminating unintended secretion of IL-6 and/or IL-8 in a drug containing an Fc fragment, such as an Fc fusion protein drug.

Drawings

Fig. 1: in CHO-K1-PD1-CTLA4 cell and human macrophage co-culture system, fc segment amino acid mutation effectively eliminates IL-8 secretion by human macrophages mediated by anti-PD-1/CTLA 4 bispecific antibodies.

Fig. 2: in CHO-K1-PD1-CTLA4 cell and human macrophage co-culture system, fc segment amino acid mutation effectively eliminates IL-6 secretion by human macrophages mediated by anti-PD-1/CTLA 4 bispecific antibodies.

Fig. 3: in the CHO-K1-PD1 cell and human macrophage co-culture system, the Fc segment amino acid mutation effectively eliminates IL-8 secretion by human macrophages mediated by anti-PD-1/CD 73 bispecific antibodies.

Fig. 4: in the CHO-K1-PD1 cell and human macrophage co-culture system, the Fc segment amino acid mutation effectively eliminates IL-6 secretion by human macrophages mediated by anti-PD-1/CD 73 bispecific antibodies.

Fig. 5: in U87-MG cell and human macrophage co-culture system, fc segment amino acid mutation effectively eliminates IL-8 secretion by human macrophages mediated by anti-PD-1/CD 73 bispecific antibody.

Fig. 6: in U87-MG cell and human macrophage co-culture system, fc segment amino acid mutation effectively eliminates IL-6 secretion by human macrophages mediated by anti-PD-1/CD 73 bispecific antibody.

Fig. 7: in the CHO-K1-PD1-LAG3 cell and human macrophage co-culture system, the Fc segment amino acid mutation effectively eliminates IL-8 secretion by human macrophages mediated by anti-PD-1/LAG 3 bispecific antibodies.

Fig. 8: in the CHO-K1-PD1-LAG3 cell and human macrophage co-culture system, the Fc segment amino acid mutation effectively eliminates IL-6 secretion by human macrophages mediated by anti-PD-1/LAG 3 bispecific antibodies.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used did not identify the manufacturer and were conventional products available commercially.

In the following experimental examples of the present invention, BALB/c mice were used purchased from the medical laboratory animal center in Guangdong province.

In the experimental example, an IgG4 subtype anti-PD-1 antibody Nivolumab (trade name, OPdivo) (Wang C et al cancer immunoRes.2014; 2 (9): 846-56.) carrying the S228P mutation and an IgG1 subtype Ipilimumab (trade name, yervoy) retaining the Fc terminal FcgammaR function were used as control antibodies, all purchased from Bai-Meissu precious corporation; igG4 subtype Relatlimab as control antibody, homemade by the chinese Kang Fang biomedical limited, lot number: 20200630.

In the following experimental examples of the present invention, the heavy chain variable region and the light chain variable region sequences of the anti-CD 73 antibody 19F3H2L3 (hG 1 WT) were used in agreement with 19F3H2L3 (hG 1 TM) in preparation example 2, and the constant region fragment used Ig gamma-1chain Cregion,ACCESSION:P01857 as the heavy chain constant region, IG KAPPA CHAIN Cregion, ACCESSION: P01834 as the light chain constant region.

In the following experimental examples of the present invention, isotype control antibodies, i.e., hIgG1 and hIgG4, were used as antibodies targeting human anti-Hen Egg Lysosomes (HEL), and the variable region sequences of these antibodies were derived from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies(Acierno et al J Mol biol.2007 published by Acierno et al; 374 (1) 130-46.) the constant region fragment of hIgG1 uses Ig gamma-1chain C region,ACCESSION:P01857 as the heavy chain constant region, IG KAPPA CHAIN C region, ACCESSION: P01834 as the light chain constant region; the hIgG4 heavy chain constant region adopts Ig gamma-4chain C region,ACCESSION:P01861.1 as a heavy chain constant region and introduces S228P mutation to improve stability, IG KAPPA CHAIN Cregion, and ACCESSION: P01834 is a light chain constant region; hIgG1, hIgG1 (DM) and hIgG4 were all prepared in the laboratory of Zhongshan Kang Fang biological medicine Co.

Preparation example 1: design and preparation of anti-PD-1/CTLA 4 bispecific antibody BiAb004 (hG 1 TM)

The structural model of the bispecific antibody BiAb004 (hG 1 TM) belongs to the Morrison model (IgG-scFv), i.e. a scFv fragment of one immunoglobulin moiety (IgG) based on the PD-1 antibody and a scFv fragment based on the anti-CTLA 4 antibody, linked in the middle by a linker fragment, linked at the C-terminus of both heavy chains of the other antibody by a linker fragment.

1. Sequence design of anti-PD-1 antibody 14C12 and humanized antibody 14C12H1L1 (hG 1 WT)

The variable region amino acid sequences and the coding nucleic acid sequences of the heavy chain and the light chain of the anti-PD-1 antibody 14C12 and the humanized antibody 14C12H1L1 (hG 1 WT) thereof are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese patent publication CN 106967172A.

(1) 14C12 heavy chain variable region sequence and light chain variable region sequence

Nucleic acid sequence encoding a 14C12 heavy chain variable region: (354 bp)

GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC(SEQ ID NO:1)

Amino acid sequence of the 14C12 heavy chain variable region: (118 aa)

EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQT PEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQM SSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA(SEQ ID NO:2)

Nucleic acid sequence encoding a 14C12 light chain variable region: (321 bp)

GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG(SEQ ID NO:3)

Amino acid sequence of the 14C12 light chain variable region: (107 aa)

DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIY YCLQYDEFPLTFGAGTKLELK(SEQ ID NO:4)

(2) Heavy and light chain variable region sequences, heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 (hG 1 WT)

Nucleic acid sequence encoding a 14C12H1L1 (hG 1 WT) heavy chain variable region (14C 12H 1V): (354 bp)

GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT(SEQ ID NO:5)

Amino acid sequence of the 14C12H1L1 (hG 1 WT) heavy chain variable region (14C 12H 1V): (118 aa)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQA PGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQM NSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS(SEQ ID NO:6)

Nucleic acid sequence encoding a 14C12H1L1 (hG 1 WT) light chain variable region (14C 12L 1V): (321 bp)

GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG(SEQ ID NO:7)

Amino acid sequence of the 14C12H1L1 (hG 1 WT) light chain variable region (14C 12L 1V): (107 aa)

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPG KSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATY YCLQYDEFPLTFGAGTKLELK(SEQ ID NO:8)

Nucleic acid sequence encoding a 14C12H1L1 (hG 1 WT) heavy chain: (1344 bp)

GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGACCTAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA(SEQ ID NO:9)

Amino acid sequence of the 14C12H1L1 (hG 1 WT) heavy chain: (448 aa)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)

Nucleic acid sequence encoding a 14C12H1L1 (hG 1 WT) light chain: (642 bp)

GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT(SEQ ID NO:11)

Amino acid sequence of the 14C12H1L1 (hG 1 WT) light chain: (214 aa)

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:12)

2. Sequence design of antibodies against CTLA4

The amino acid sequences and the coding nucleic acid sequences of the heavy chain and the light chain of the anti-CTLA 4 antibody 4G10 and the humanized antibody 4G10H3L3 are respectively identical to those of 4G10 and 4G10H3L3 in the Chinese patent publication CN 106967172A.

(1) Heavy chain variable region sequence and light chain variable region sequence of 4G10

Nucleic acid sequence encoding the 4G10 heavy chain variable region: (372 bp)

CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAGTTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT(SEQ ID NO:13)

Amino acid sequence of the 4G10 heavy chain variable region: (124 aa)

QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY(SEQ ID NO:14)

Nucleic acid sequence encoding a 4G10 light chain variable region: (378 bp)

CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC(SEQ ID NO:15)

Amino acid sequence of the 4G10 light chain variable region: (126 aa)

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC(SEQ ID NO:16)

(2) Heavy chain variable region sequence and light chain variable region sequence of humanized monoclonal antibody 4G10H3L3

Nucleic acid sequence encoding the 4G10H3L3 heavy chain variable region (4G 10H 3V): (345 bp)

CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAACTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA(SEQ ID NO:17)

Amino acid sequence of the 4G10H3L3 heavy chain variable region (4G 10H 3V): (115 aa)

QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMEL SRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:18) Nucleic acid sequence encoding a 4G10H3L3 light chain variable region (4G 10L 3V): (327 bp)

CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG(SEQ ID NO:19)

Amino acid sequence of the 4G10H3L3 light chain variable region (4G 10L 3V): (109 aa)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFGGGTKLTVL(SEQ ID NO:20)

3. Sequence design of bispecific antibody BiAb004 (M)

The structural pattern of bispecific antibody BiAb004 (M) belongs to the Morrison pattern (IgG-scFv), i.e. a scFv fragment of one IgG antibody linked to the other by a linker fragment at the C-terminus of both heavy chains, the design composition of the heavy and light chains of which is shown in table 1 below.

Table 1: sequence design of BiAb004 (M)

In table 1 above:

The amino acid sequence of Linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 29)

In addition, in the scFv fragment of the BiAb004 (M) antibody in table 1, the 4G10H3V (M) and 4G10L3V (M) are obtained by mutating individual amino acids in the framework regions thereof on the basis of 4G10H3V and 4G10L3V, so that the structure of the antibody is effectively optimized and the effectiveness thereof is improved.

(1) 4G10H3V (M): (115 aa, amino acid mutation sites based on the heavy chain variable region 4G10H3V of 4G10H3L3 are underlined)

QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQCLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMEL SRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:30)

(4) 4G10L3V (M): (110 aa, amino acid mutation sites based on the light chain variable region 4G10H3L3 of 4G10L3 are underlined)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFGCGTKLTVLR(SEQ ID NO:31)

In order to distinguish from the following mutated antibody BiAb004 (hG 1 TM), biAb004 (M) is referred to as "wild type" in the examples of the present invention, also referred to as BiAb004 (hG 1 WT). BiAb004 (hG 1 WT) uses Ig gamma-1chain C region,ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin portion, IG KAPPA CHAIN C region, ACCESSION: P01834 as the light chain constant region of the immunoglobulin portion.

4. Non-variable region amino acid mutation design based on humanized bispecific antibody BiAb004 (hG 1 WT)

Based on BiAb004 (hG 1 WT) obtained above, the present inventors have obtained BiAb004 (hG 1 TM) by introducing a leucine to alanine point mutation (L234A) at the 234 th position, a leucine to alanine point mutation (L235A) at the 235 th position and a glycine to alanine point mutation (G237A) at the 237 th position of the heavy chain according to the EU numbering system. The remaining amino acid sequence was identical to BiAb004 (hG 1 WT).

Preparation example 2: design and preparation of anti-PD-1/CD 73 bispecific antibody NTPDV (hG 1 TM)

The structural pattern of bispecific antibody NTPDV (hG 1 TM) belongs to the Morrison pattern (IgG-scFv), i.e. a scFv fragment of one IgG antibody linked to the other by a linker fragment at the C-terminus of both heavy chains, the design composition of the heavy and light chains of which is shown in table 2 below. NTPDV2 (hG 1 TM) uses Ig gamma-1chain C region,ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin portion and IG KAPPA CHAIN Cregion, ACCESSION: P01834 as the light chain constant region of the immunoglobulin portion, and 3 mutations were made according to the EU numbering system on this basis: L234A, L a and G237A.

Table 2: NTPDV2 sequence design of 2 (hG 1 TM)

In table 2 above:

the amino acid sequence of Linker is shown in the previous SEQ ID NO. 29.

The amino acid sequence of 14C12H1V is shown in SEQ ID NO. 6.

The amino acid sequence of 14C12L1V is shown in SEQ ID NO. 8.

The nucleic acid sequence encoding the heavy chain variable region of 19F3H2 (hG 1 TM) is as follows (363 bp), the CDR-encoding regions being underlined:

CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC(SEQ ID NO:21)

the amino acid sequence of the heavy chain variable region of 19F3H2 (hG 1 TM) is as follows (121 aa), the CDR regions being underlined:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS(SEQ ID NO:22)

the nucleic acid sequence encoding the heavy chain of 19F3H2 (hG 1. TM.) is as follows (1353 bp):

CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGCgcctccacaaaggggcccagcgtgtttcctctcgccccctcctccaaaagcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtcaccgtgagctggaatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggactctattccctgtccagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtcaaccacaagccctccaacaccaaggtggacaagaaagtggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctcccgaaGCTGCTggagCccctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaacccctgaagtcacctgtgtcgtcgtggatgtcagccatgaggaccccgaggtgaaattcaactggtatgtcgatggcgtcgaggtgcacaacgccaaaaccaagcccagggaggaacagtacaactccacctacagggtggtgtccgtgctgacagtcctccaccaggactggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggctctccctgcccccattgagaagaccatcagcaaggccaaaggccaacccagggagccccaggtctatacactgcctccctccagggacgaactcaccaagaaccaggtgtccctgacctgcctggtcaagggcttttatcccagcgacatcgccgtcgagtgggagtccaacggacagcccgagaataactacaagaccacccctcctgtcctcgactccgacggctccttcttcctgtacagcaaactgaccgtcgataaatctaggtggcagcagggcaacgtgttctcttgttccgtgatgcatgaagcactgcacaaccattatacccagaagtctctgagcctgtcccccggcaag(SEQ ID NO:23)

The amino acid sequence of the heavy chain of 19F3H2 (hG 1. TM.) is as follows (451 aa), the CDR regions are underlined:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)

The nucleic acid sequence (339 bp) encoding the light chain variable region of 19F3L3 was as follows:

GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ ID NO:25)

the amino acid sequence (113 aa) of the light chain variable region of 19F3L3 is as follows:

DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWY QQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQHYDTPYTFGGGTKLEIK(SEQ ID NO:26)

19F3L3 as the light chain of the immunoglobulin portion in NTPDV (hG 1. TM.) the nucleic acid sequence (660 bp) encoding 19F3L3 is as follows:

GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCTGAGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT(SEQ ID NO:27)

19F3L3 as light chain of the immunoglobulin part in NTPDV (hG 1 TM) the amino acid sequence is as follows (220 aa), wherein the CDR regions are underlined and bolded:

DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:28)

preparation example 3: design and preparation of anti-PD-1/LAG 3 bispecific antibody Bs-PL022B (hG 1 TM)

The structural pattern of bispecific antibody Bs-PL022B (hG 1 TM) belongs to the Morrison pattern (IgG-scFv), i.e. the scFv fragment of one IgG antibody is linked to the other by a linker fragment at the C-terminus of both heavy chains, the design composition of the heavy and light chains of which is shown in table 3 below. Bs-PL022B (hG 1 TM) uses Ig gamma-1chain C region,ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin portion and IG KAPPA CHAIN Cregion, ACCESSION: P01834 as the light chain constant region of the immunoglobulin portion, and 3 mutations are made according to the EU numbering system on this basis: L234A, L235A, L a.

Table 3: sequence design of Bs-PL022B (hG 1 TM)

In table 3 above:

the amino acid sequence of Linker is shown in the previous SEQ ID NO. 29: GGGGSGGGGSGGGGGGSGGGGS

Nucleic acid sequence (360 bp) of the heavy chain variable region H7v of H7L 8:

CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAACACTGGTTACAGTGAGCTCC(SEQ ID NO:56)

amino acid sequence (120 aa) of heavy chain variable region H7v of H7L 8:

QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQP PGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRS VTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS(SEQ ID NO:57)

nucleic acid sequence of the light chain variable region L8v of H7L8 (321 bp):

GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:58)

Amino acid sequence of the light chain variable region L8v of H7L8 (107 aa):

EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQ APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPITFGQGTNLEIK(SEQ ID NO:59)

amino acid sequence of heavy chain of immunoglobulin part in Bs-PL022B (hG 1 TM):

QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:60)

nucleic acid sequence of heavy chain of immunoglobulin part in Bs-PL022B (hG 1 TM):

CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAACACTGGTTACAGTGAGCTCCGCCTCCACCAAGGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAAGCTGCTGGAGCCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAACTGACCGTCGATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCCGTGATGCATGAAGCACTGCACAACCATTATACCCAGAAGTCTCTGAGCCTGTCCCCCGGCAAG(SEQ ID NO:61)

amino acid sequence of light chain of immunoglobulin part in Bs-PL022B (hG 1 TM):

EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:62)

Nucleic acid sequence of the light chain of the immunoglobulin part in Bs-PL022B (hG 1 TM):

GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCTGAGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT(SEQ ID NO:63)

Individual amino acids in their framework regions (light chain) were mutated based on 14C12H1L1 to give 14C12H1L1 (M).

Heavy chain variable region 14C12H1 (M) _V of 14C12H1L1 (M):

Identical to the heavy chain variable region 14C12H1 (hG 1 WT) of 14C12H1L1, i.e., the amino acid sequence is shown in SEQ ID NO. 6.

Light chain variable region 14C12L1 (M) _V of 14C12H1L1 (M): (108 aa, amino acid sequence mutation sites based on the light chain variable region of 14C12H1L1 (hG 1 WT) are underlined

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQ KPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQP EDMATYYCLQYDEFPLTFGAGTKLELKR(SEQ ID NO:64)

Experimental example 1: fc-segment mutations are effective in eliminating IL-8 and IL-6 secretion mediated by anti-PD-1/CTLA 4 bispecific antibodies, immune checkpoint inhibitors

1. Experimental materials:

human peripheral macrophages (Human peripheral monocyte derived macrophage, HPMM) are induced by human peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELLS, PBMCs). PBMCs used in this study were all prepared separately from the biological medicine company, kang Fang in the chinese mountain and informed consent was provided.

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat# 17-1440-03); RPMI 1640 (Gibco, cat# 22400-105); CHO-K1-PD1-CTLA4 cells (constructed by the company Kang Fang biomedical limited in zhongshan); FBS (Fetal Bovine Serum, excel bio, cat# FSP 500); human IFN-gamma protein (sinobio, cat# 11725-HNAS-100); LPS (Lipopolysaccharides) lipopolysaccharide (sigma, cat# L4391); 96 well cell culture plates (Corning, cat# 3599).

2. The experimental method comprises the following steps:

Healthy human Peripheral Blood Mononuclear Cells (PBMC) were isolated according to the protocol of Ficoll-Paque ^TM Plus reagent and resuspended in 1640 medium with 2% FBS and placed in a 5% CO ₂ cell incubator at 37 ℃. After 2h the supernatant was removed and adherent cells were washed 2 times with PBS and induced by addition of 1640 complete medium (with 10% FBS) and 100ng/mL human M-CSF for 7 days. The fluid was changed on days 3 and 5 and M-CSF was supplemented to induce HPMM. After HPMM induction was completed, HPMM cells were collected to 1 ten thousand per well, IFN-gamma (working concentration 50 ng/mL) was added and plated in 96-well plates, and the total volume of each well was 100. Mu.L. Collecting CHO-K1 cells expressing human PD-1 and CTLA4, namely CHO-K1-PD1-CTLA4 cells, and regulating the cell number to 3 ten thousand/100 mu L/hole; the antibodies (working concentrations: 25nM, 2.5nM, 0.25 nM) were diluted in complete 1640 medium, 100. Mu.L of antibody dilution was added per well according to the experimental design, mixed well, and isotype control wells were designed. Lipopolysaccharide (LPS) was used as a positive control drug, and the concentration was adjusted to 100ng/mL from complete medium in the experiment. The cell plates were placed in an incubator and incubated for 24h. After centrifugation at 1200rpm for 5min, the supernatant was collected and assayed for IL-8, IL-6 secretion using the daceae kit.

In this example, CHO-K1-PD1-CTLA4 cells co-cultured with HPMM as target cells induced HPMM activation, and after activated HPMM linked to target cells by antibody Fab, the Fc fragment of the antibody acted on fcγr on HPMM, causing HPMM to secrete cytokines.

3. Experimental results

As shown in fig. 1 and 2.

The results show that anti-PD-1/CTLA 4 bispecific antibodies (BiAb 004 (hG 1 WT)) carrying an Fc fragment mutation are able to effectively eliminate IL-6 and/or IL-8 secretion by immune cells compared to wild type IgG1 subtype antibodies, nivolumab of the IgG4 subtype and Ipilimumab of the IgG1 subtype, which introduce an S228P mutation to improve stability, administered in combination, with wild type Fc fragment.

Experimental example 2: fc-segment mutations are effective in eliminating IL-8 and IL-6 secretion mediated by bispecific antibodies to the bispecific immune checkpoint inhibitor PD-1/CD73

HPMM was induced by PBMC. PBMCs used in this study were all prepared separately from the biological medicine company, kang Fang in the chinese mountain and informed consent was provided.

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat# 17-1440-03); RPMI 1640 (Gibco, cat# 22400-105); CHO-K1-PD1 cells (constructed by the biological medicine company of zhongshan Kang Fang); U87-MG cells (cells derived from ATCC, available from the former advanced technology Co., ltd., beijing); FBS (Fetal Bovine Serum, excel bio, cat# FSP 500); human IFN-gamma protein (sinobio, cat# 11725-HNAS-100); LPS (Lipopolysaccharides) lipopolysaccharide (sigma, cat# L4391); 96 well cell culture plates (Corning, cat# 3599).

Healthy human PBMC were isolated according to the protocol of the isolate Ficoll-Paque ^TM Plus reagent and resuspended in 1640 medium with 2% FBS and placed in a 5% CO ₂ cell incubator at 37 ℃. After 2h the supernatant was removed and adherent cells were washed 2 times with PBS and induced by addition of 1640 complete medium (with 10% FBS) and 100ng/mL human M-CSF for 7 days. The fluid was changed on days 3 and 5 and M-CSF was supplemented to induce HPMM. After induction is completed on day HPMM, the cells are collected, the concentration of the cells is adjusted to 10 ten thousand/mL by using a complete culture medium, the cells are split into 96-well plates, and recombinant human IFN-gamma (50 ng/mL) is added, and the cells are placed in an incubator for incubation for 24 hours. After 24h, the log phase of CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 were collected and the concentration was adjusted to 30 ten thousand/mL with complete medium after resuspension. The antibodies were diluted to a working concentration of 25nM, 2.5nM, 0.25nM in complete medium. Isotype control antibodies and blank controls were designed simultaneously. The supernatant from the 96-well plate was removed, and the CHO-K1-PD1 or U87-MG cell suspension and antibody (final volume 200. Mu.L) were added and mixed well and incubated in an incubator for 24 hours. After centrifugation at 500Xg for 5min, the supernatant was collected and assayed for IL-8, IL-6 secretion using the daceae kit. LPS was used as a positive control drug and the concentration was adjusted to 100ng/mL from complete medium in the experiment.

In this example, CHO-K1-PD1 and U87-MG cells were co-cultured with HPMM as target cells to induce HPMM activation, and after activated HPMM was linked to target cells by antibody Fab, the Fc fragment of the antibody acted on fcγr on HPMM, causing HPMM to secrete cytokines.

3. Experimental results

As shown in fig. 3 to 6.

The results show that anti-PD-1/CD 73 bispecific antibodies carrying an Fc segment mutation are effective in eliminating IL-6 and/or IL-8 secretion by immune cells compared to the wild type IgG1 subtype of PD-1 antibody or CD73 antibody.

In addition, anti-PD-1/CD 73 bispecific antibodies carrying an Fc segment mutation are also able to effectively eliminate secretion of IL-6 and/or IL-8 by immune cells as compared to wild type bispecific antibody NTPDV (hG 1 WT) which does not carry an Fc segment mutation.

Experimental example 3: fc-segment mutations are effective in eliminating IL-8 and IL-6 secretion mediated by anti-PD-1/LAG 3 bispecific antibodies, immune checkpoint inhibitors

1. Experimental materials:

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat# 17-1440-02); RPMI 1640 (Gibco, cat# 22400-105); CHO-K1-PD1-LAG3 cells (constructed by the biosciences company Kang Fang, zhongshan); FBS (Fetal Bovine Serum, excel bio, cat# FSP 500); human IFN-gamma protein (sinobio, cat# 11725-HNAS-100); LPS (Lipopolysaccharides, sigma, cat# L6529); 96 well cell culture plates (Corning, cat# 3599).

2. The experimental method comprises the following steps:

Healthy human Peripheral Blood Mononuclear Cells (PBMC) were isolated according to the protocol of Ficoll-Paque ^TM Plus reagent and resuspended in 1640 medium with 2% FBS and placed in a 5% CO ₂ cell incubator at 37 ℃. After 2h the supernatant was removed and adherent cells were washed 2 times with PBS and induced by addition of 1640 complete medium (with 10% FBS) and 100ng/mL human M-CSF for 7 days. The fluid was changed on days 3 and 5 and M-CSF was supplemented to induce HPMM. After HPMM induction was completed, the cells were collected, HPMM cells were adjusted to 1 ten thousand per well, IFN-gamma (working concentration 50 ng/mL) was added and plated in 96-well plates for 120 wells total, and the total volume per well was 100. Mu.L. Collecting CHO-K1 cells expressing human PD-1 and LAG3, namely CHO-K1-PD1-LAG3 cells, and regulating the cell number to 3 ten thousand/100 mu L/hole; the antibodies (working concentrations: 25nM, 2.5nM, 0.25 nM) were diluted in 1640 complete medium, 100. Mu.L of antibody dilution was added per well according to the experimental design, mixed well, and isotype control wells were designed. Lipopolysaccharide (LPS) was used as a positive control drug, and the concentration was adjusted to 100ng/mL from complete medium in the experiment. The cell plates were placed in an incubator and incubated for 24h. After taking out the cell plate and centrifuging at 1200rpm for 5min, the supernatant was collected and assayed for IL-8 and IL-6 secretion using the daceae kit.

In this example, co-culturing CHO-K1-PD1-LAG3 cells as target cells with HPMM induces HPMM activation, and after activated HPMM links to target cells by antibody Fab, the Fc fragment of the antibody acts on fcγr on HPMM, causing HPMM to secrete cytokines.

3. Experimental results

As shown in fig. 7 and 8.

The results showed that anti-PD-1/LAG 3 bispecific antibodies (Bs-PL 022B (hG 1 TM)) carrying an Fc segment mutation are able to effectively eliminate secretion of IL-6 and/or IL-8 by immune cells compared to wild type IgG1 subtype antibodies, relatlimab of the Nivolumab, igG subtype of IgG4 subtype, which introduces an S228P mutation to improve stability, anti-PD-1 antibody 14C12H1L1 (hG 1 WT) carrying no Fc segment mutation, and anti-LAG 3 antibody H7L8 (hG 1 WT) carrying a wild type Fc segment.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method of reducing the level of IL-8 and/or IL-6 secreted by an immune cell mediated by a drug comprising an immunoglobulin Fc fragment, comprising the steps of:

the immunoglobulin Fc fragment was subjected to the following mutations according to the EU numbering system:

L234A and L235A;

L234A and G237A;

L235A and G237A;

Or alternatively

L234A, L a and G237A;

wherein the method is a method of non-therapeutic interest;

Wherein the immunoglobulin Fc fragment-containing drug comprises an antibody, which is a bispecific antibody,

Wherein the bispecific antibody targets LAG3 and PD-1, comprising:

a first protein functional region targeting LAG3, and

A second protein functional region targeting PD-1;

Wherein:

The first protein functional region is immunoglobulin, and the second protein functional region is a single-chain antibody; wherein, the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 50-52 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 53-55 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 32-34 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 35-37 respectively;

Or alternatively

The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; wherein, the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs 32-34 respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs 35-37 respectively; and the heavy chain variable region of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs 50-52 respectively, and the light chain variable region of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs 53-55 respectively;

The immunoglobulin is human IgG; and

2. The method of claim 1, wherein the drug comprising an immunoglobulin Fc fragment further comprises one or more pharmaceutically acceptable excipients.

3. The method of claim 1, wherein,

Or alternatively

4. A method according to claim 3, wherein the bispecific antibody is selected from any one of (1) - (6) as follows:

(1)

(2)

(3)

(4)

(5)

(6)

5. The method of any one of claims 1 to 4, wherein the heavy chain constant region of the immunoglobulin is selected from the group consisting of a heavy chain constant region of human IgG1, igG2, igG3 or IgG4, and the light chain constant region of the immunoglobulin is selected from the group consisting of a light chain constant region of human IgG1, igG2, igG3 or IgG 4.

6. The method of claim 5, wherein the heavy chain constant region of the immunoglobulin is a human Ig gamma-1chain C region or a human Ig gamma-4chain C region and the light chain constant region of the immunoglobulin is a human IG KAPPA CHAIN C region.

7. The method of any one of claims 1 to 4, wherein the immune cells are human immune cells.

8. The method of claim 7, wherein the immune cells are human macrophages.

9. The method of any one of claims 1 to 4, wherein the method is a method for pharmaceutical purposes.

10. A method of increasing the effectiveness and/or safety of a medicament comprising an immunoglobulin Fc fragment, wherein the level of IL-8 and/or IL-6 secreted by a medicament-mediated immune cell comprising an immunoglobulin Fc fragment is reduced by the method of any one of claims 1 to 9.