CN115073588A

CN115073588A - Method for improving safety of medicine containing immunoglobulin Fc fragment

Info

Publication number: CN115073588A
Application number: CN202210243178.3A
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: 2022-09-20
Also published as: WO2022188867A1

Abstract

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

Description

Method for improving safety of medicine containing 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 increasing the safety and/or effectiveness of the drug. In particular, the invention relates to a method of reducing or blocking the levels of IL-8 and/or IL-6 secreted by immune cells mediated by an agent containing an Fc fragment of immunoglobulin, such as an antibody or Fc fusion protein.

Background

Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells or somatic cells that recognize the Fc region of antibodies that mediate an immune response. After the Fab region of the antibody recognizes the antigen, the Fc region of the antibody binds to Fc receptors on immune cells (e.g., killer cells) and activates effector cells.

Fc receptors are mainly classified into three types, Fc γ R, Fc α R and Fc ∈ R, according to the type of antibody recognized by the Fc receptor and the difference of cells expressing the Fc receptor, and Fc γ R is classified into four subtypes, Fc γ RI (also referred to as CD64), Fc γ RII (also referred to as CD32), Fc γ RIII (also referred to as CD16), and FcRn (also referred to as Neonatal Fc receptor). Wherein the Fc gamma RI, the Fc gamma RII and the Fc gamma RIII are closely related to ADCC effect. Fc γ RIII is the most predominant molecule mediating ADCC, with two subtypes of Fc γ RIIIa and Fc γ RIIIb being highly homologous in different cell types, with the high affinity Fc γ RIIIa subtype arising from the presence of single-nucleotide stem polymorphism (SNP) sites in the Fc γ RIIIa population, referred to as the two subtypes Fc γ RIIIa _ V158 and low affinity Fc γ RIIIa _ F158, respectively. The Fc gamma RI has higher affinity to the Fc region of IgG and participates in the ADCC process; the Fc gamma RII comprises three subtypes of Fc gamma RIIa, Fc gamma RIIb and Fc gamma RIic (also called CD32a, CD32b and CD32c respectively), wherein the Fc gamma RIIa has ADCC activity; two subtypes due to single nucleotide mutations exist in the human population, designated Fc γ RIIa _ H131 and Fc γ RIIa _ R131(Hogarth PM, Pietesz GA.2012, Nature Review Drug Discovery,11(4): 311-.

The IgG family comprises four members, IgG1, IgG2, IgG3, and IgG4, which differ in their affinity for Fc γ Rs due to amino acid differences in the fragment crystallizable (Fc) region of the heavy chain constant region. IgG1 is the most abundant subtype in humans and also the most abundant subtype used in monoclonal antibody drugs, IgG1 is able to bind various Fc γ Rs. IgG2 had the weakest affinity for Fc γ Rs, but IgG2 was still able to bind Fc γ RIIa. IgG3 bound most strongly to Fc γ Rs. The IgG4 molecule binds weakly to Fc γ Rs other than Fc γ RI. IgG4 antibody subtypes are unstable, and hinge region cleavage easily occurs, which leads to Fab-arm exchange, and formation of half-molecule and bispecific, functionally monovalent antibodies (Aalberse R.C.et al.Clin.exp.Allergy.2009; 39(4): 469-77.); the introduction of the S228P mutation in the heavy chain hinge region of the IgG4 antibody stabilized the IgG4 molecule, preventing the formation of half molecules (Shirley J Peters et al J Biol chem.201213; 287(29): 242533.).

ADCR (antibody-dependent cytokine release) refers to antibody-dependent cytokine release, in which the Fab fragment of an antibody binds to an epitope of a tumor cell, the Fc fragment cross-binds to an effector cell surface Fc Receptor (Fc Receptor, FcR), and activates effector cells by cross-linking, resulting in massive secretion of cytokines by activated effector cells, such as secreted release of IL (interleukin) -1, IL-6, IL-8, IL-10, MCP (monocyte chemotactic protein) -1, and the like, wherein IL-6 is a major inflammatory mediator. These cytokines can reduce the efficacy of immunotherapy and increase immune-related side effects, which can seriously lead to multiple organ failure and death.

Interleukin-8 (IL-8) is a Chemotactic cytokine (Chemotactic cytokines) belonging to the CXC-alpha subfamily (also known as CXCL-8). In normal human bodies, the antigen is mainly secreted by monocytes, immune cells, epithelial cells and the like and participates in inflammation and immune defense reaction in the bodies; the receptor (CXCR) is a dimeric glycoprotein composed of two subunits of 59 and 67kDa, belongs to the superfamily of G protein-coupled receptors, and shares two subtypes, namely CXCR1 and CXCR 2. IL-8 plays an important role in the proliferation of normal cells and tumor cells, and especially plays an important role in promoting the occurrence and development of tumors. Research shows that IL-8 can promote the generation of tumors; tumor cells themselves can 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 indispensable in the tumor microenvironment.

IL-8 as a proinflammatory factor is closely related to the occurrence and development of tumors. During the process of inducing malignant transformation of non-kidney cancer cells by methylarsinate (methyarsonate), the expression of IL-8 gene is increased, IL-8 gene silencing can obviously inhibit the growth of transplanted tumor in mice, and in addition, the reduction of 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 tumor (Escudero-Lourdes C et al: morphology and applied pharmacology, 2012, 258: 10-18.). Inoue et al found that IL-8 induced malignant and invasive growth of non-neoplastic bladder cell lines (233JP), while the incidence of malignant transformation of 233JP cells was significantly reduced in IL-8 knockdown mice (Inoue K et al cancer Res,2000, 60(8): 2290-2299.). Furthermore, in prostate Cancer, IL-8 can promote the development of castration-resistant prostate Cancer (CRPC) in patients (Chen K et al. Cancer research,2015, 75(10): 1992-; gene silencing of IL-8 or its receptor induces cell cycle arrest in tumor cells and inhibits tumor proliferation (Singh RK, Lokeshwar BL. molecul Cancer,2009,8: 57.). The above studies indicate that the level of IL-8 is closely related to the development and development of tumors. Further studies (Mian BM et al. Clin Cancer Res, 2003,9(8):3167-3175.) have shown that IL-8 can be a novel target for tumor therapy. In a tumor model of bladder cancer, the growth of tumor can be obviously inhibited by using an anti-IL-8 antibody.

IL-6 is rapidly produced primarily by macrophages, responds to pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), and cures damaged tissue by removing infectious agents, inducing acute phases and an immune response, and serves as a protective effect.

IL-6 also responds to include Toll-like receptor (TLR) ligands and proinflammatory cytokines such as IL-1 and TNF- α. In the context of infected lesions, IL-6 is produced by stimulation of TLRs on monocytes and macrophages, each TLR recognizing the 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 noninfectious inflammation such as burns and wounds, the level 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 1 (HMGB1), heat shock proteins, and S100 molecules, 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 injury, high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells, thereby inhibiting myocardial function, and even can cause "cytokine storm" to produce severe acute systemic inflammatory responses. Cytokine storms are a lethal complication and adverse reaction in viral infections, tumor immunotherapy, and the like.

Immune-related adverse reactions are a common and dangerous adverse reaction in Immune Checkpoint Inhibitor (ICI) antitumor therapy (Spain L et al cancer Treat rev.2016; 44: 51-60.). In recent years, immune checkpoint inhibitors have had great success in tumor immunotherapy, but also have resulted in a completely new toxicity profile due to off-target effects. In which severe immune-related adverse events (irAE), especially of major organs including heart, lung and brain, may be life threatening (Bergqvist V, et al. cancer immune system, 2017; 66(5): 581-. Data have shown that ICI can induce off-target effects by 4 mechanisms, including direct binding to immunodetection site molecules expressed on the surface of normal cells, activating complement hypersensitivity; normal tissues present with homologous antigens/epitopes to tumor cells; generating an autoantibody; increasing The levels of pro-inflammatory cytokines, such as IL-6 and The like (Martins F et al, The Lancet Oncology,20(1), e 54-e 64.).

Currently, anti-IL-6 therapy, such as Tolizumab, a recombinant humanized anti-IL-6R monoclonal antibody, has 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), e 54-e 64.).

Fc fusion protein drugs, such as IL-2-Fc fusion protein, have been proved to be used for treating tumor at present, however, because of the great toxic effect of the Fc fusion protein drugs, for example, the IL-2-Fc fusion protein can cause lethal capillary leakage and induce proliferation of immunosuppressive Treg cells, and influence the antitumor activity of the cells, if the Fc fragment can further induce the immune cells to secrete IL-8 and/or IL-6, the antitumor effectiveness and safety of the cells are significantly influenced. These all limit their clinical applications.

In conclusion, the Fc fusion protein drug and the immune checkpoint inhibitor, especially the antibody drug targeting the immune checkpoint and the Fc fusion protein drug taking the cytokine, the chemokine and the ligand thereof as action mechanisms inhibit the effect of inducing the immune cells to secrete IL-8 and/or IL-6, and have great significance for improving the effectiveness and/or safety of the drug.

Disclosure of Invention

The present inventors have conducted intensive research and creative efforts to modify the Fc-terminus of an anti-dual immune checkpoint inhibitor (anti-PD-1/CTLA 4 bispecific antibody, anti-PD-1/CD 73 bispecific antibody, anti-PD-1/LAG 3 bispecific antibody, etc., or an Fc fusion protein with immunomodulatory biological activity, such as a fusion protein of IL-2 and Fc), so as to effectively reduce or eliminate the activity of an immune checkpoint therapeutic antibody or fusion protein mediated or induced undesired immune cells to secrete IL-6 and/or IL-8, thereby increasing the safety and/or effectiveness of the immune checkpoint inhibitor and fusion protein drug. The following invention is thus provided:

one aspect of the present 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

L234A, L235A and G237A.

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

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

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

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

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

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

It will be understood by those skilled in the art that a drug containing an immunoglobulin Fc fragment is actually a pharmaceutical composition when it contains one or more pharmaceutically acceptable excipients. Can be prepared into various dosage forms, such as injection, etc., according to the skill of those 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 LAG 3.

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

targeting a first protein functional region of PD-1, and

targeting a second protein domain of CTLA 4;

wherein the first protein functional region is immunoglobulin and the second protein functional region is 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 content of the first and second substances,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:38-40, respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:41-43, respectively;

alternatively, the first and second electrodes may be,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:32-34 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:35-37 respectively;

wherein the content of the first and second substances,

the immunoglobulin is human IgG;

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

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 variable region of the light chain 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;

alternatively, the first and second electrodes may be,

the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from 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 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 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) to (18) as follows:

(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:

targeting a first protein functional region of CD73, and

a second protein functional region targeted to PD-1;

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

wherein the content of the first and second substances,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:32-34 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:35-37 respectively;

alternatively, the first and second electrodes may be,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:44-46 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:47-49 respectively;

wherein the content of the first and second substances,

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 variable region of the light chain of the immunoglobulin has an amino acid sequence 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 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 SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64;

alternatively, the first and second electrodes may be,

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 variable region of the light chain 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 variable region of the light chain 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) to (6) as follows:

(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 comprises:

targeting a first protein functional region of LAG3, and

a second protein functional region targeted to PD-1;

wherein the content of the first and second substances,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:32-34 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:35-37 respectively;

alternatively, the first and second electrodes may be,

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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:50-52, respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:53-55, respectively;

wherein the content of the first and second substances,

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 tail 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 57; and the amino acid sequence of the variable region of the light chain 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 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 SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64;

alternatively, the first and second electrodes may be,

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 variable region of the light chain 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 57; and the amino acid sequence of the variable region of the light chain 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 immunoglobulin heavy chain constant region of the bispecific antibody is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, and the immunoglobulin light chain constant region of the bispecific antibody is selected from the light chain constant regions of human IgG1, IgG2, IgG3, or IgG 4;

preferably, the immunoglobulin heavy chain constant region of the bispecific antibody is human Ig gamma-1chain C region or human Ig gamma-4chain C region, and the immunoglobulin light chain constant region of the bispecific antibody is 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 immunoglobulin heavy chain constant region of the bispecific antibody is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4, and the immunoglobulin light chain constant region of the bispecific antibody is selected from the light chain constant regions 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

L234A, L235A and G237A.

Another example is:

in some embodiments of the invention, the method wherein the immunoglobulin heavy chain constant region of the bispecific antibody is human Ig gamma-1chain C region or human Ig gamma-4chain C region, and the immunoglobulin light chain constant region 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

L234A, L235A and G237A.

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

In some embodiments of the invention, the method is a method for non-therapeutic purposes.

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

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

Another aspect of the present invention relates to a method for 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 immune cells mediated or induced by the drug comprising an immunoglobulin Fc fragment is reduced by the method of any one of the present invention. In some embodiments of the invention, the method is a pharmaceutical method.

Those skilled in the art know 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) (CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3, CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR 3; which are named by Kabat et al, see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol.1-3, NIH Publication 91-3242, Bethesda Md).

The amino acid sequences of the CDR regions of the antibody sequences to which the invention relates are analyzed by technical means well 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 3 CDR regions of its heavy chain variable region is identical to that of 14C 12.

The variable region in the light chain has the same amino acid sequence as 14C12 in the 3 CDR regions.

(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 3 CDR regions of its heavy chain variable region is identical to that of 4G 10.

The amino acid sequence of 3 CDR regions of its 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;

the variable region of its heavy chain has the same amino acid sequence as 4G10 in its 3 CDR regions.

(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, scientific and technical terms used herein have meanings commonly understood by those skilled in the art, unless otherwise specified. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, when referring to the amino acid sequence of a 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 of PD-1, PD-1ECD, or a fragment comprising PD-1 ECD; also included are fusion proteins of PD-1ECD, such as a fragment fused to an Fc protein fragment (mFc or hFc) of a 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 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 a sequence fragment of the PD-1 protein is described, 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 segment of CTLA4 CTLA4 ECD or a segment comprising CTLA4 ECD; also included are fusion proteins of CTLA4 ECD, such as 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 into the amino acid sequence of CTLA4 protein without affecting its biological function. Thus, in the present invention, the term "CTLA 4 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when sequence fragments of CTLA4 protein are described, they include CTLA4 sequence fragments, as well as 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, CD73 ECD or a fragment comprising CD73 ECD; also included are fusion proteins of CD73 ECD, such as a fragment fused to an Fc protein fragment (mFc or hFc) of a 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 into the amino acid sequence of the CD73 protein without affecting its biological function. Thus, in the present invention, the term "CD 73 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the CD73 protein, it includes a fragment of the CD73 sequence, and also includes a fragment of the corresponding sequence in natural or artificial variants thereof.

As used herein, when referring to the amino acid sequence of the LAG3 protein, it includes, but is not limited to, the full length of the LAG3 protein (NCBI Genebank ID: NP-002277.4), or the extracellular fragment LAG3 ECD of LAG3 or a fragment comprising the LAG3 ECD; also included are fusion proteins of LAG3 ECD, such as a fragment fused to an Fc protein fragment (mFc or hFc) of a 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 the LAG3 protein without affecting its biological function. Thus, in the present invention, the term "LAG 3 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of LAG3 protein, it includes a fragment of LAG3 sequence, and also includes a corresponding sequence fragment 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 antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected 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 (CH1, 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 the antibody may mediate the binding of the 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). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed 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 the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; 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 antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

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 high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature,256:495,1975), but can also be obtained using recombinant DNA technology (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 a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions 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 amino acid residues of the Framework Regions (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by 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: 522525 (1986); reichmann et al, Nature,332: 323329 (1988); presta, curr.op. struct.biol.,2: 593596 (1992); and Clark, immunol. today 21: 3973 (2000).

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be the case that it is in a natural environment that has been altered, or that the substance has been isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of other impurities which do not interfere with the activity of the substance, either mixed with artificial or synthetic substances.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of conferring expression on 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 element it carries is 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 Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (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 contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, 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 the antigen against which it is directed. In certain embodiments, an antibody that specifically binds an antigen (or an antibody specific for an antigen) means that the antibody binds less than about 10 ^-5 M, e.g. less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、 10 ^-9 M or 10 ^-10 M or less binds to the antigen with an affinity (KD).

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

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" 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. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is 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, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as 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, a desired effect. For example, a prophylactically effective amount (e.g., tumor) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., tumor); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an 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, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "completely eliminated" refers to the absence of binding signal or very low binding signal as detected by existing instrumentation (e.g., Fortebio Octet molecular interaction instrumentation). In one embodiment of the invention, very low binding signal means a binding signal below 0.1 nm.

In the present invention, the term "Fusion Protein (FP)" refers to a protein product obtained by expressing two or more genes encoding functional proteins by purposefully linking the genes together and expressing the desired proteins, by linking the coding regions of two or more genes end-to-end under artificial conditions, and controlling the resulting genes with the same regulatory sequence.

In the present invention, the term "immunoglobulin (Ig) fusion protein" refers to a recombinant protein having two domains as described above, which is expressed in eukaryotic or prokaryotic cells by linking a gene of interest to an Ig partial fragment gene at the gene level. Proteins of interest are classified into two broad categories, according to their association with different fragments of Ig: one class is fab (fv) fusion proteins; another 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, such as a soluble ligand (or receptor) molecule capable of binding to an endogenous receptor (or ligand) or other active substance (e.g., cytokine) requiring an extended half-life, with an Fc fragment using genetic engineering techniques. The Fc fusion protein mainly combines a bioactive protein with a hinge region of Ig and a CH2 and CH3 region.

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

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

In the present invention, the term "chemokines" is a specific class of cytokines, low molecular weight proteins that influence leukocyte chemotaxis and other cellular behaviors, and play an important role in the middle of the inflammatory response.

In the present invention, letters before the site indicate amino acids before mutation, and letters after the site indicate amino acids after mutation, if not otherwise specified.

Advantageous effects of the invention

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

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

(2) the present invention can also exert an effect of effectively eliminating the secretion of IL-6 and/or IL-8, which is not intended, in a drug containing an Fc fragment, such as an Fc fusion protein drug.

Drawings

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

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

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

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

FIG. 5: in a co-culture system of U87-MG cells and human macrophages, the Fc segment amino acid mutation effectively eliminates IL-8 secretion by human macrophages mediated by an anti-PD-1/CD 73 bispecific antibody.

FIG. 6: in a co-culture system of U87-MG cells and human macrophages, the Fc segment amino acid mutation effectively eliminates IL-6 secretion by human macrophages mediated by an 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 of human macrophage cells mediated by the anti-PD-1/LAG 3 bispecific antibody.

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 of human macrophage cells mediated by the anti-PD-1/LAG 3 bispecific antibody.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The examples do not indicate any particular technique or condition, according to the techniques or conditions described in the literature of the art (for example, see molecular cloning, A laboratory Manual, third edition, scientific Press, ed. by J. SammBruker et al, Huang Pentang et al) or according to the instructions for the product. The reagents or instruments used are not indicated by the manufacturer, but are conventional products available on the market.

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

In the experimental examples, Nivolumab (trade name Opdivo) which is an IgG4 subtype anti-PD-1 antibody carrying the S228P mutation (Wang C et al cancer Immunol Res.2014; 2(9):846-56.) and Iplilimumab (trade name Yervoy) which is an IgG1 subtype retaining Fc γ R function at the Fc terminal were used as control antibodies and were purchased from Michelia Baikh; IgG4 subtype Relatlimab, a control antibody, was manufactured by zhongshan kang bio-pharmaceuticals, ltd, lot No.: 20200630.

in the following experimental examples of the present invention, the sequences of the heavy chain variable region and the light chain variable region of 19F3H2L3(hG1WT) of the anti-CD 73 antibody used were identical to those of 19F3H2L3(hG1TM) in preparation example 2, and the constant region fragments used were 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.

In the following experimental examples of the present invention, the isotype control antibodies, hIgG1 and hIgG4, were antibodies targeted to human anti-Hen Egg Lysosome (HEL), and the variable region sequences of these antibodies were derived from Affinity information and specificity of the Fv domain of anti-protein antibodies (Acierno et al J Mol biol 2007; 374 (130-46)), the constant region fragment of hIgG1 was 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 heavy chain constant region of hIgG4 adopts Ig gamma-4chain C region, ACCESSION, P01861.1 as heavy chain constant region and introduces S228P mutation to improve stability, Ig kappa chain C region, ACCESSION, P01834 as light chain constant region; hIgG1, hIgG1(DM) and hIgG4 were all made in the laboratory of Zhongshan Kangfang biomedical Co., Ltd.

Preparation example 1: design and preparation of anti-PD-1/CTLA 4 bispecific antibody BiAb004(hG1TM)

The structural pattern of the bispecific antibody BiAb004(hG1TM) belongs to the Morrison pattern (IgG-scFv), i.e. the C-termini of both heavy chains of one immunoglobulin part (IgG) antibody are linked to the scFv fragment of the other antibody by a linking fragment, the immunoglobulin part being based on the PD-1 antibody and the scFv fragment being based on the anti-CTLA 4 antibody, linked in between by the linking fragment.

1. Sequence design of anti-PD-1 antibody 14C12 and humanized antibody 14C12H1L1(hG1WT) thereof

The amino acid sequences of the variable regions of the heavy chain and the light chain of the anti-PD-1 antibody 14C12 and the humanized antibody 14C12H1L1(hG1WT) thereof, and the encoding nucleic acid sequences are completely identical to 14C12 and 14C12H1L1 in Chinese patent publication CN 106967172A, respectively.

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

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

GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAA GCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCT TCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCC CTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGC GGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTC ACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAG ATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGT GCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACA GGGCACCCTGGTGACAGTCTCTGCC(SEQ ID NO:1)

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

EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQT PEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQM SSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA(SEQ ID NO:2)

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

GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCT AGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCA GGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGG CAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGC AGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACA TGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGA CCTTTGGAGCAGGCACAAAACTGGAACTGAAG(SEQ ID NO: 3)

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

DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIY YCLQYDEFPLTFGAGTKLELK(SEQ ID NO:4)

(2) Heavy chain and light chain variable region sequences, heavy chain and light chain sequences of humanized monoclonal antibody 14C12H1L1(hG1WT)

Nucleic acid sequence encoding the variable region of the heavy chain of 14C12H1L1(hG1WT) (14C12H 1V): (354bp)

GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCA GCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGAT TCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCAC CAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGC GGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTT CACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATT GTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGG CAGGGAACCCTGGTGACAGTCTCTAGT(SEQ ID NO:5)

Amino acid sequence of heavy chain variable region (14C12H1V) of 14C12H1L1(hG1 WT): (118aa)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQA PGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQM NSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS(SEQ ID NO:6)

Nucleic acid sequence encoding the variable region of the 14C12H1L1(hG1WT) light chain (14C12L 1V): (321bp)

GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCC TCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCA GGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGG GAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGAC AGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACA TGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGA CCTTTGGCGCCGGGACAAAACTGGAGCTGAAG(SEQ ID NO: 7)

Amino acid sequence of 14C12H1L1(hG1WT) light chain variable region (14C12L 1V): (107aa)

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPG KSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATY YCLQYDEFPLTFGAGTKLELK(SEQ ID NO:8)

Nucleic acid sequence encoding the 14C12H1L1(hG1WT) heavy chain: (1344bp)

GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCA GCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGAT TCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCAC CAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGC GGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTT CACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATT GTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGG CAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGG ACCTAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAG CGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACT TCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTG ACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGC GGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGC AGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATC CTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGA ACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACC CAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTG TGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAAT TCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAA ACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGT GGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACG GCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCT GCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACC CAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACG AACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGG GCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACG GACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCG ACTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGG ACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGC GTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGC CTGTCCCTGAGCCCCGGCAAA(SEQ ID NO:9)

Amino acid sequence of the 14C12H1L1(hG1WT) heavy chain: (448aa)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQA PGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQM NSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK(SEQ ID NO:10)

Nucleic acid sequence encoding 14C12H1L1(hG1WT) light chain: (642bp)

GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCC TCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCA GGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGG GAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGAC AGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACA TGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGA CCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTG GCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAG CTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAAT TTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAA CGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAAC AGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGA CTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCAT GCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAG TCTTTTAACCGGGGCGAATGT(SEQ ID NO:11)

Amino acid sequence of 14C12H1L1(hG1WT) light chain: (214aa)

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPG KSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATY YCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:12)

2. Sequence design of anti-CTLA 4 antibodies

The amino acid sequences and coding nucleic acid sequences of the heavy chain and light chain of anti-CTLA 4 antibody 4G10 and its humanized antibody 4G10H3L3 are the same as 4G10 and 4G10H3L3 in chinese patent publication CN 106967172a, respectively.

(1)4G10 heavy chain variable region sequence and light chain variable region sequence

Nucleic acid sequence encoding the variable region of the heavy chain of 4G 10: (372bp)

CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAA GCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTA CTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCA TGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAA TAATATTACTAACTACAACCAGAAGTTCATGGGCAAGGCCAC ATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACT CCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGC AAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO:13)

Amino acid sequence of the heavy chain variable region of 4G 10: (124aa)

QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQS HGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMEL LRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO:14)

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

CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCA CCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGG GCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAA CCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAAC CGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATT GGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGA GGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCA TTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCC AGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAAT TCTGC(SEQ ID NO:15)

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

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEK PDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEA IYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO:16)

(2) The heavy chain variable region sequence and the light chain variable region sequence of humanized monoclonal antibody 4G10H3L3

Nucleic acid sequence encoding the variable region of the heavy chain of 4G10H3L3 (4G10H 3V): (345bp)

CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAA ACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGGT ACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCC CTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTAC AACAACATCACTAACTACGCACAGAAGTTCCAGGGGAGAGT GACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGA ACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTG CGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACAC TGGTGACTGTCAGCGCA(SEQ ID NO:17)

Amino acid sequence of the heavy chain variable region of 4G10H3L3 (4G10H 3V): (115aa)

QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMEL SRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:18)

Nucleic acid sequence encoding the variable region of the light chain of 4G10H3L3 (4G10L 3V): (327bp)

CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCT CCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGG GGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAA GCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACA ACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTG CTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCC TGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAA CCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (SEQ ID NO:19)

Amino acid sequence of 4G10H3L3 light chain variable region (4G10L 3V): (109aa)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFGGGTKLTVL(SEQ ID NO:20)

3. Sequence design of bispecific antibody BiAb004(M)

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

Table 1: sequence design of BiAb004(M)

In table 1 above:

the amino acid sequence of Linker is GGGGSGGGGSGGSGGGGS (SEQ ID NO:29)

In addition, 4G10H3V (M) and 4G10L3V (M) in the scFv fragment of BiAb004(M) antibody in table 1 above are obtained by mutating individual amino acids in the framework region of 4G10H3V and 4G10L3V, which effectively optimizes the structure of the antibody and improves its effectiveness.

(1)4G10H3V (M): (115aa, 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): (110aa, amino acid mutation sites based on the light chain variable region 4G10H3L3, 4G10L3V, underlined)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFGCGTKLTVLR(SEQ ID NO:31)

In order to distinguish from the mutated antibody BiAb004(hG1TM) below, in the present example BiAb004(M) was taken as "wild-type", also referred to as BiAb004(hG1 WT). BiAb004(hG1WT) used Ig gamma-1chain C region, ACCESSION: P01857 as the heavy chain constant region of the immunoglobulin moiety and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region of the immunoglobulin moiety.

4. Design of non-variable region amino acid mutations based on humanized bispecific antibody BiAb004(hG1WT)

Based on the BiAb004(hG1WT) obtained above, the present inventors obtained BiAb004(hG1TM) by introducing a leucine to alanine point mutation at position 234 (L234A), a leucine to alanine point mutation at position 235 (L235A) and a glycine to alanine point mutation at position 237 (G237A) in accordance with the EU numbering system. The remaining amino acid sequence was identical to BiAb004(hG1 WT).

Preparation example 2: design and preparation of anti-PD-1/CD 73 bispecific antibody NTPDV2(hG1TM)

The structural pattern of bispecific antibody NTPDV2(hG1TM) belongs to the Morrison pattern (IgG-scFv), i.e. a scFv fragment of one IgG antibody is linked at the C-terminus of both heavy chains of the other antibody by a linking fragment, the design composition of its heavy and light chains is as in table 2 below. NTPDV2(hG1TM) used Ig gamma-1chain C region, ACCESSION: P01857 as the heavy chain constant region of the immunoglobulin moiety and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region of the immunoglobulin moiety, and based thereon 3 mutations were made according to the EU numbering system: L234A, L235A and G237A.

Table 2: sequence design of NTPDV2(hG1TM)

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 preceded by SEQ ID NO 6.

The amino acid sequence of 14C12L1V is preceded by SEQ ID NO 8.

The nucleic acid sequence encoding the heavy chain variable region of 19F3H2(hG1TM) is as follows (363bp), the CDR-encoding regions are underlined:

CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAG CCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCA GGACAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGTGACC CTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTG AGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCC GGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGG GGCCAGGGCACCACACTGACCGTGTCTAGC(SEQ ID NO:21)

the amino acid sequence of the heavy chain variable region of 19F3H2(hG1TM) is as follows (121aa), with the CDR regions underlined:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQ APGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYME LSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO:22)

the nucleic acid sequence encoding the heavy chain of 19F3H2(hG1TM) is as follows (1353 bp):

CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAG CCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTAC TCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCA GGACAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAAT GCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGTGACC CTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTG AGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCC CGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGG GGCCAGGGCACCACACTGACCGTGTCTAGCgcctccacaaaggggccca gcgtgtttcctctcgccccctcctccaaaagcaccagcggaggaaccgctgctctcggatgtctggtga aggactacttccctgaacccgtcaccgtgagctggaatagcggcgctctgacaagcggagtccataca ttccctgctgtgctgcaaagcagcggactctattccctgtccagcgtcgtcacagtgcccagcagcagc ctgggcacccagacctacatctgtaacgtcaaccacaagccctccaacaccaaggtggacaagaaag tggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctcccgaaGCTGCTgga gCccctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaacccctgaagtc acctgtgtcgtcgtggatgtcagccatgaggaccccgaggtgaaattcaactggtatgtcgatggcgtc gaggtgcacaacgccaaaaccaagcccagggaggaacagtacaactccacctacagggtggtgtcc gtgctgacagtcctccaccaggactggctgaacggcaaggagtacaagtgcaaggtgtccaacaagg ctctccctgcccccattgagaagaccatcagcaaggccaaaggccaacccagggagccccaggtcta tacactgcctccctccagggacgaactcaccaagaaccaggtgtccctgacctgcctggtcaagggct tttatcccagcgacatcgccgtcgagtgggagtccaacggacagcccgagaataactacaagaccac ccctcctgtcctcgactccgacggctccttcttcctgtacagcaaactgaccgtcgataaatctaggtgg cagcagggcaacgtgttctcttgttccgtgatgcatgaagcactgcacaaccattatacccagaagtct ctgagcctgtcccccggcaag(SEQ ID NO:23)

the amino acid sequence of the heavy chain of 19F3H2(hG1TM) is as follows (451aa), with CDR regions underlined:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQA PGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELS SLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK(SEQ ID NO:24)

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

GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGT CTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTA TCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGG CAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCT GCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCAGCACT ACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGAT CAAG(SEQ ID NO:25)

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

DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWY QQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQHYDTPYTFGGGTKLEIK(SEQ ID NO:26)

19F3L3 As the light chain of the immunoglobulin part in NTPDV2(hG1TM), the nucleic acid sequence (660bp) encoding 19F3L3 is as follows:

GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTG TCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCA GTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTG GTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCT ACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTC TCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGC TCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCA GCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCT GGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTTT TCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGT GGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGT GCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCAC AGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATA GTCTGAGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAA AGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGGGCTG AGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT (SEQ ID NO:27)

19F3L3 as the light chain of the immunoglobulin part of NTPDV2(hG1TM) has the following amino acid sequence (220aa), wherein the CDR regions are shown underlined and bolded:

preparation example 3: design and preparation of anti-PD-1/LAG 3 bispecific antibody Bs-PL022B (hG1TM)

The structural pattern of bispecific antibody Bs-PL022B (hG1TM) is in Morrison mode (IgG-scFv), i.e. the scFv fragments of one IgG antibody are linked at the C-termini of both heavy chains of the other antibody by a linker fragment, the design composition of their heavy and light chains is as in table 3 below. Bs-PL022B (hG1TM) used Ig gamma-1chain C region, ACCESSION: P01857 as the heavy chain constant region of the immunoglobulin moiety and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region of the immunoglobulin moiety, and based thereon 3 mutations were made according to the EU numbering system: L234A, L235A, L237A.

Table 3: sequence design of Bs-PL022B (hG1TM)

In table 3 above:

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

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

CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAA ACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCG GATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCC CTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGG GGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACA CTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTG AGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCC TTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGG GGCCAGGGAACACTGGTTACAGTGAGCTCC(SEQ ID NO:56)

amino acid sequence of heavy chain variable region H7v of H7L8 (120 aa):

QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQP PGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRS VTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS(SEQ ID NO:57)

the nucleic acid sequence (321bp) of the light chain variable region L8v of H7L 8:

GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTG TCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCA GACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTG GCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGG CCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGA ACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGAT TTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATC ACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO: 58)

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

EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQ APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPITFGQGTNLEIK(SEQID NO:59)

amino acid sequence of heavy chain of immunoglobulin fraction in Bs-PL022B (hG1 TM):

QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQP PGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRS VTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK(SEQ ID NO:60)

the nucleic acid sequence of the heavy chain of the immunoglobulin fraction in Bs-PL022B (hG1 TM):

CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAA ACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCG GATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCC CTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGG GGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACA CTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTG AGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCC TTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGG GGCCAGGGAACACTGGTTACAGTGAGCTCCGCCTCCACCAA GGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCAC CAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACT ACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTC TGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCA GCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCA GCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACA AGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAA TCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCC GAAGCTGCTGGAGCCCCTAGCGTCTTCCTCTTTCCTCCCAAA CCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACC TGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAA ATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAA AACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGG TGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCC TGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAAC CCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGAC GAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAG GGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAAC GGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTC GACTCCGACGGCTCCTTCTTCCTGTACAGCAAACTGACCGTC GATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCC GTGATGCATGAAGCACTGCACAACCATTATACCCAGAAGTCT CTGAGCCTGTCCCCCGGCAAG(SEQ ID NO:61)

Bs-PL022B (hG1TM) amino acid sequence of the light chain of the immunoglobulin fraction:

EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQ APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:62)

the nucleic acid sequence of the light chain of the immunoglobulin fraction in Bs-PL022B (hG1 TM):

GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTG TCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCA GACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTG GCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGG CCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGA ACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGAT TTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATC ACATTCGGACAGGGCACAAATCTGGAGATCAAGCGTACGGT GGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCA GCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAA TTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCGATAA CGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAAC AGGACTCCAAGGATTCTACCTATAGTCTGAGCTCCACTCTGA CCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCT GTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAAT CCTTCAACAGAGGCGAATGT(SEQ ID NO:63)

individual amino acids in the framework region (light chain) were mutated based on 14C12H1L1 to give 14C12H1L1 (M).

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

The same as the heavy chain variable region 14C12H1(hG1WT) of 14C12H1L1, namely the amino acid sequence is shown in SEQ ID NO: 6.

Light chain variable region of 14C12H1L1(M) 14C12L1(M) _V : (108aa, amino acid sequence mutation sites based on the light chain variable region of 14C12H1L1(hG1WT) are underlined)

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQ KPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQP EDMATYYCLQYDEFPLTFGAGTKLELKR(SEQ ID NO:64)

Experimental example 1: fc segment mutation can effectively eliminate bispecific antibody of anti-PD-1/CTLA 4 by immune checkpoint inhibitor Mediated secretion of IL-8 and IL-6

1. Experimental materials:

human peripheral macrophages (HPMMs) are induced from Human Peripheral Blood Mononuclear Cells (PBMCs). PBMCs used in this study were all prepared separately in zhongshan kang biomedical limited and with the informed consent of the providers.

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat # 17-1440-03); RPMI 1640(Gibco, cat # 22400-105); CHO-K1-PD1-CTLA4 cell (constructed by Zhongshan Kangfang biological medicine Co., Ltd.); FBS (Fetal bone Serum, Excell bio, cat # FSP 500); human IFN-gamma protein (sinobio, cat # 11725-HNAS-100); lps (lipopolysaccharides), lipopolysaccharides (sigma,the goods number is: l4391); 96-well cell culture plates (Kangning, cat # 3599).

2. The experimental method comprises the following steps:

according to the separation liquid Ficoll-Paque ^TM Plus reagent Instructions healthy human Peripheral Blood Mononuclear Cells (PBMC) were isolated and resuspended in 1640 medium containing 2% FBS at 37 deg.C, 5% CO ₂ Placing in a cell culture box. After 2h the supernatant was removed, adherent cells were washed 2 times with PBS and induced for 7 days by addition of 1640 complete medium (containing 10% FBS) and 100ng/mL human M-CSF. Fluid was changed and supplemented with M-CSF to induce HPMM on days 3 and 5. After the completion of HPMM induction, the number of HPMM cells was adjusted to 1 ten thousand per well, and IFN-. gamma.was added (working concentration: 50ng/mL) to the well of a 96-well plate in a total volume of 100. mu.L per well. Collecting CHO-K1 cells expressing human PD-1 and CTLA4, i.e., CHO-K1-PD1-CTLA4 cells, and adjusting the number of the cells to 3 ten thousand/100 mu L/hole; the antibody (working concentration: 25nM, 2.5nM, 0.25nM) was diluted with complete 1640 medium, 100. mu.L of antibody diluent 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 are all placed in a culture box for incubation for 24 h. Centrifuging at 1200rpm for 5min, collecting supernatant, and detecting IL-8 and IL-6 secretion amount with Dake kit.

In this example, the co-culture of CHO-K1-PD1-CTLA4 cells as target cells with HPMM induces the activation of HPMM, and after the activated HPMM is linked to the target cells by antibody Fab, the Fc fragment of the antibody interacts with Fc γ R on HPMM to cause the cytokine secretion by HPMM.

3. Results of the experiment

As shown in fig. 1 and 2.

The results show that the combined administration of Nivolumab of IgG4 subtype and Ipilimumab of IgG1 subtype, which introduced S228P mutation to improve stability, and anti-PD-1/CTLA 4 bispecific antibody with wild-type Fc fragment (BiAb004(hG1WT)), and anti-PD-1/CTLA 4 bispecific antibody carrying Fc fragment mutation (BiAb004(hG1TM)) can effectively eliminate IL-6 and/or IL-8 secretion from immune cells, compared to wild-type IgG1 subtype antibody.

Experimental example 2: fc segment mutation can effectively eliminate dual specificity immunityCheckpoint inhibitor PD-1/CD73 bispecific Antibody-mediated secretion of IL-8 and IL-6

HPMM was induced from PBMC. PBMCs used in this study were all prepared separately in zhongshan kang biomedical limited and with the informed consent of the providers.

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat # 17-1440-03); RPMI 1640(Gibco, cat # 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Kangfang biomedicine Co., Ltd.); U87-MG cells (cells from ATCC, original technology of China limited, Beijing); FBS (Fetal bone Serum, Excell bio, cat # FSP 500); human IFN-gamma protein (sinobio, cat # 11725-HNAS-100); LPS (Lipopolysaccharides), lipopolysaccharide (sigma, cat # L4391); 96-well cell culture plates (Kangning, cat # 3599).

According to the separating medium Ficoll-Paque ^TM Plus reagent Instructions healthy human PBMC were isolated and resuspended in 1640 medium containing 2% FBS at 37 deg.C, 5% CO ₂ Placing in a cell culture box. After 2h the supernatant was removed, adherent cells were washed 2 times with PBS and induced for 7 days by addition of 1640 complete medium (containing 10% FBS) and 100ng/mL human M-CSF. Fluid was changed and supplemented with M-CSF to induce HPMM on days 3 and 5. After completion of HPMM induction on day 7, the cells were collected, adjusted to a concentration of 10 ten thousand/mL with the use of the whole medium, dispensed into 96-well plates, and recombinant human IFN-. gamma.was added (50ng/mL), and left in an incubator for 24 hours. CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 were collected at log phase after 24h, and the concentration was adjusted to 30 ten thousand/mL with complete medium after resuspension. The antibody was diluted with complete medium to working concentrations of 25nM, 2.5nM, 0.25 nM. And designing an isotype control antibody and a blank control at the same time. The supernatant in the 96-well plate is removed, and CHO-K1-PD1 or U87-MG cell suspension and antibody (final volume 200. mu.L) are added and mixed evenly, and the mixture is placed in an incubator and incubated for 24 h. Centrifuging at 500Xg for 5min, collecting supernatant, and detecting IL-8 and IL-6 secretion amount with Dake as reagent kit. LPS was used as a positive control drug and was adjusted to a concentration of 100ng/mL from complete medium in the experiment.

In this example, the co-culture of CHO-K1-PD1 and U87-MG cells as target cells with HPMM induces the activation of HPMM, and after the activated HPMM is linked to the target cells by antibody Fab chain, the Fc fragment of the antibody acts on Fc γ R on HPMM, causing cytokine secretion by HPMM.

3. Results of the experiment

As shown in fig. 3-6.

The results show that the anti-PD-1/CD 73 bispecific antibody carrying an Fc fragment mutation can effectively eliminate IL-6 and/or IL-8 secretion from immune cells compared to the wild-type IgG1 subtype PD-1 antibody or CD73 antibody.

Furthermore, the anti-PD-1/CD 73 bispecific antibody carrying the Fc fragment mutation was also able to effectively eliminate IL-6 and/or IL-8 secretion by immune cells compared to the wild-type bispecific antibody NTPDV2(hG1WT) which does not carry the Fc fragment mutation.

Experimental example 3: fc segment mutation can effectively eliminate medium of anti-PD-1/LAG 3 bispecific antibody by immune checkpoint inhibitor Directed secretion of IL-8 and IL-6

1. Experimental materials:

Ficoll-Paque ^TM PLUS lymphocyte isolate (GE, cat # 17-1440-02); RPMI 1640(Gibco, cat # 22400-105); CHO-K1-PD1-LAG3 cell (constructed by Zhongshan Kangfang biomedicine Co., Ltd.); FBS (Fetal bone Serum, Excell bio, cat # FSP 500); human IFN-gamma protein (sinobio, cat # 11725-HNAS-100); LPS (Lipopolysaccharides, sigma, cat # L6529); 96-well cell culture plates (Kangning, cat # 3599).

2. The experimental method comprises the following steps:

according to the separation liquid Ficoll-Paque ^TM Plus reagent Instructions healthy human Peripheral Blood Mononuclear Cells (PBMC) were isolated and resuspended in 1640 medium containing 2% FBS at 37 deg.C, 5% CO ₂ Cell culturePlacing in a breeding box. After 2h the supernatant was removed, adherent cells were washed 2 times with PBS and induced for 7 days by addition of 1640 complete medium (containing 10% FBS) and 100ng/mL human M-CSF. Fluid was changed and supplemented with M-CSF to induce HPMM on days 3 and 5. After the HPMM induction was completed, the number of HPMM cells was adjusted to 1 ten thousand per well, and IFN-. gamma.was added (working concentration: 50ng/mL) and the mixture was plated in 96-well plates for a total of 120 wells, each well having a total volume of 100. mu.L. Collecting CHO-K1 cells expressing human PD-1 and LAG3, i.e., CHO-K1-PD1-LAG3 cells, and adjusting the number of the cells to 3 ten thousand per 100 mu L per well; the antibody (working concentration: 25nM, 2.5nM, 0.25nM) was diluted with 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 all placed in an incubator and incubated for 24 h. The cell plate was removed and centrifuged at 1200rpm for 5min, and the supernatant was collected and assayed for the secretion of IL-8 and IL-6 using Dake's kit.

In this example, the co-culture of CHO-K1-PD1-LAG3 cells as target cells with HPMM induces the activation of HPMM, which is linked to the target cells via antibody Fab, and the Fc fragment of the antibody interacts with Fc γ R on HPMM to cause the secretion of cytokine from HPMM.

3. Results of the experiment

As shown in fig. 7and 8.

The results showed that the anti-PD-1/LAG 3 bispecific antibody (Bs-PL022B (hG1TM)) carrying the Fc fragment mutation can effectively eliminate the secretion of IL-6 and/or IL-8 by immune cells compared to wild-type IgG1 subtype antibody, Nivolumab of IgG4 subtype having improved stability by introducing the S228P mutation, Relatimab of IgG1 subtype, anti-PD-1 antibody 14C12H1L1(hG1WT) carrying no Fc fragment mutation, and anti-LAG 3 antibody H7L8(hG1WT) carrying the wild-type Fc fragment.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Zhongshan Kangfang biomedical Co., Ltd

<120> method for improving safety of immunoglobulin Fc fragment-containing drugs

<130> IDC200505

<150> CN202110270671.X

<151> 2021-03-12

<160> 64

<170> PatentIn version 3.5

<210> 1

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the variable region of the heavy chain of 14C12

<400> 1

gaggtcaaac tggtggagag cggcggcggg ctggtgaagc ccggcgggtc actgaaactg 60

agctgcgccg cttccggctt cgcctttagc tcctacgaca tgtcatgggt gaggcagacc 120

cctgagaagc gcctggaatg ggtcgctact atcagcggag gcgggcgata cacctactat 180

cctgactctg tcaaagggag attcacaatt agtcgggata acgccagaaa tactctgtat 240

ctgcagatgt ctagtctgcg gtccgaggat acagctctgt actattgtgc aaaccggtac 300

ggcgaagcat ggtttgccta ttggggacag ggcaccctgg tgacagtctc tgcc 354

<210> 2

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain variable region of 14C12

<400> 2

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 3

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the variable region of the 14C12 light chain

<400> 3

gacattaaga tgacacagtc cccttcctca atgtacgcta gcctgggcga gcgagtgacc 60

ttcacatgca aagcatccca ggacatcaac acatacctgt cttggtttca gcagaagcca 120

ggcaaaagcc ccaagaccct gatctaccgg gccaatagac tggtggacgg ggtccccagc 180

agattctccg gatctggcag tgggcaggat tactccctga ccatcagctc cctggagtat 240

gaagacatgg gcatctacta ttgcctgcag tatgatgagt tccctctgac ctttggagca 300

ggcacaaaac tggaactgaa g 321

<210> 4

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 14C12 light chain variable region amino acid sequence

<400> 4

Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 5

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the variable region of the heavy chain of 14C12H1L1(hG1WT) (14C12H1V)

<400> 5

gaagtgcagc tggtcgagtc tgggggaggg ctggtgcagc ccggcgggtc actgcgactg 60

agctgcgcag cttccggatt cgcctttagc tcctacgaca tgtcctgggt gcgacaggca 120

ccaggaaagg gactggattg ggtcgctact atctcaggag gcgggagata cacctactat 180

cctgacagcg tcaagggccg gttcacaatc tctagagata acagtaagaa caatctgtat 240

ctgcagatga acagcctgag ggctgaggac accgcactgt actattgtgc caaccgctac 300

ggggaagcat ggtttgccta ttgggggcag ggaaccctgg tgacagtctc tagt 354

<210> 6

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain variable region (14C12H1V) of 14C12H1L1(hG1WT)

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 7

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the variable region of the 14C12H1L1(hG1WT) light chain (14C12L1V)

<400> 7

gacattcaga tgactcagag cccctcctcc atgtccgcct ctgtgggcga cagggtcacc 60

ttcacatgcc gcgctagtca ggatatcaac acctacctga gctggtttca gcagaagcca 120

gggaaaagcc ccaagacact gatctaccgg gctaatagac tggtgtctgg agtcccaagt 180

cggttcagtg gctcagggag cggacaggac tacactctga ccatcagctc cctgcagcct 240

gaggacatgg caacctacta ttgcctgcag tatgatgagt tcccactgac ctttggcgcc 300

gggacaaaac tggagctgaa g 321

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of light chain variable region (14C12L1V) of 14C12H1L1(hG1WT)

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 9

<211> 1344

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the 14C12H1L1(hG1WT) heavy chain

<400> 9

gaagtgcagc tggtcgagtc tgggggaggg ctggtgcagc ccggcgggtc actgcgactg 60

agctgcgcag cttccggatt cgcctttagc tcctacgaca tgtcctgggt gcgacaggca 120

ccaggaaagg gactggattg ggtcgctact atctcaggag gcgggagata cacctactat 180

cctgacagcg tcaagggccg gttcacaatc tctagagata acagtaagaa caatctgtat 240

ctgcagatga acagcctgag ggctgaggac accgcactgt actattgtgc caaccgctac 300

ggggaagcat ggtttgccta ttgggggcag ggaaccctgg tgacagtctc tagtgccagc 360

accaaaggac ctagcgtgtt tcctctcgcc ccctcctcca aaagcaccag cggaggaacc 420

gctgctctcg gatgtctggt gaaggactac ttccctgaac ccgtcaccgt gagctggaat 480

agcggcgctc tgacaagcgg agtccataca ttccctgctg tgctgcaaag cagcggactc 540

tattccctgt ccagcgtcgt cacagtgccc agcagcagcc tgggcaccca gacctacatc 600

tgtaacgtca accacaagcc ctccaacacc aaggtggaca agaaagtgga gcccaaatcc 660

tgcgacaaga cacacacctg tcccccctgt cctgctcccg aactcctcgg aggccctagc 720

gtcttcctct ttcctcccaa acccaaggac accctcatga tcagcagaac ccctgaagtc 780

acctgtgtcg tcgtggatgt cagccatgag gaccccgagg tgaaattcaa ctggtatgtc 840

gatggcgtcg aggtgcacaa cgccaaaacc aagcccaggg aggaacagta caactccacc 900

tacagggtgg tgtccgtgct gacagtcctc caccaggact ggctgaacgg caaggagtac 960

aagtgcaagg tgtccaacaa ggctctccct gcccccattg agaagaccat cagcaaggcc 1020

aaaggccaac ccagggagcc ccaggtctat acactgcctc cctccaggga cgaactcacc 1080

aagaaccagg tgtccctgac ctgcctggtc aagggctttt atcccagcga catcgccgtc 1140

gagtgggagt ccaacggaca gcccgagaat aactacaaga ccacccctcc tgtcctcgac 1200

tccgacggct ccttcttcct gtacagcaag ctgaccgtgg acaaaagcag gtggcagcag 1260

ggaaacgtgt tctcctgcag cgtgatgcac gaagccctcc acaaccacta cacccagaaa 1320

agcctgtccc tgagccccgg caaa 1344

<210> 10

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> 14C12H1L1(hG1WT) heavy chain amino acid sequence

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 642

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding 14C12H1L1(hG1WT) light chain

<400> 11

gacattcaga tgactcagag cccctcctcc atgtccgcct ctgtgggcga cagggtcacc 60

ttcacatgcc gcgctagtca ggatatcaac acctacctga gctggtttca gcagaagcca 120

gggaaaagcc ccaagacact gatctaccgg gctaatagac tggtgtctgg agtcccaagt 180

cggttcagtg gctcagggag cggacaggac tacactctga ccatcagctc cctgcagcct 240

gaggacatgg caacctacta ttgcctgcag tatgatgagt tcccactgac ctttggcgcc 300

gggacaaaac tggagctgaa gcgaactgtg gccgctccct ccgtcttcat ttttccccct 360

tctgacgaac agctgaaatc aggcacagcc agcgtggtct gtctgctgaa caatttctac 420

cctagagagg caaaagtgca gtggaaggtc gataacgccc tgcagtccgg caacagccag 480

gagagtgtga ctgaacagga ctcaaaagat agcacctatt ccctgtctag tacactgact 540

ctgtccaagg ctgattacga gaagcacaaa gtgtatgcat gcgaagtgac acatcaggga 600

ctgtcaagcc ccgtgactaa gtcttttaac cggggcgaat gt 642

<210> 12

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> 14C12H1L1(hG1WT) light chain amino acid sequence

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 13

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding 4G10 heavy chain variable region

<400> 13

caggtcaagc tgcaggagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60

tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc 120

catggaaaga accttgaatg gattggactt attaatcctt acaataatat tactaactac 180

aaccagaagt tcatgggcaa ggccacattt actgtagaca agtcatccag cacagcctac 240

atggaactcc tcagactgac atctgaagac tctggagtct atttctgtgc aagactcgac 300

tataggtctt attggggcca agggactctg gtcactgtct ctgcagccaa aacgacaccc 360

ccatctgtct at 372

<210> 14

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 4G10 heavy chain variable region

<400> 14

Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Asn Ile Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Met Gly Lys Ala Thr Phe Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Arg Leu Thr Ser Glu Asp Ser Gly Val Tyr Phe Cys

85 90 95

Ala Arg Leu Asp Tyr Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120

<210> 15

<211> 378

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding 4G10 light chain variable region

<400> 15

caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60

acttgtcgct caagtactgg ggctgttaca actagtaact ttgccaactg ggtccaagaa 120

aaaccagatc atttattcac tagtctaata ggtggtacca acaaccgagc tccaggtgtt 180

cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca 240

cagactgagg atgaggcaat atatttctgt gctctatggt acagcaacca ttgggtgttc 300

ggtggaggaa ccaaactgac tgtcctaggc cagcccaagt cttcgccatc agtcaccctg 360

tttcaagggc aattctgc 378

<210> 16

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of light chain variable region of 4G10

<400> 16

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Phe Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Ser

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ser Ser Pro Ser Val Thr Leu Phe Gln Gly Gln Phe Cys

115 120 125

<210> 17

<211> 345

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the variable region of the heavy chain of 4G10H3L3 (4G10H3V)

<400> 17

caggtgcagc tggtcgagtc tggggccgaa gtgaagaaac ccggcgcctc agtgaaggtc 60

agctgcaagg ccagcgggta cagtttcact ggatatacca tgaactgggt ccgacaggcc 120

cctggccagg ggctggagtg gatcggcctg attaaccctt acaacaacat cactaactac 180

gcacagaagt tccaggggag agtgaccttt acagtggaca ccagcatttc cacagcctac 240

atggaactgt cccggctgag atctgacgat acaggcgtgt acttctgcgc taggctggat 300

taccgcagct attggggaca gggcacactg gtgactgtca gcgca 345

<210> 18

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain variable region of 4G10H3L3 (4G10H3V)

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Asn Ile Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg Leu Asp Tyr Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 19

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding 4G10H3L3 light chain variable region (4G10L3V)

<400> 19

caggctgtcg tcactcagga accttcactg accgtgtctc ctggcgggac tgtcaccctg 60

acatgcggca gctccacagg ggccgtgacc acaagtaact tcccaaattg ggtccagcag 120

aagccaggac aggctccccg gagtctgatc ggaggcacca acaacaaggc cagctggaca 180

cccgcacggt tcagcggcag cctgctgggc ggcaaggccg ctctgacaat tagcggagcc 240

cagcctgagg acgaagccga gtactattgc gctctgtggt actccaacca ctgggtgttc 300

ggcggcggca ccaagctgac tgtgctg 327

<210> 20

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of light chain variable region of 4G10H3L3 (4G10L3V)

<400> 20

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Phe Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser

35 40 45

Leu Ile Gly Gly Thr Asn Asn Lys Ala Ser Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 21

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the heavy chain variable region of 19F3H2(hG1TM)

<400> 21

caggtgcagc tggtgcagtc tggagcagag gtggtgaagc caggagcctc tgtgaaggtg 60

agctgtaagg ccagcggcta ctccttcacc ggctatacaa tgaactgggt gaggcaggca 120

ccaggacaga atctggagtg gatcggcctg atcaaccctt acaatgccgg cacctcttat 180

aaccagaagt ttcagggcaa ggtgaccctg acagtggaca agtccacctc tacagcctac 240

atggagctga gctccctgcg gagcgaggat acagccgtgt actattgcgc ccggtccgag 300

tacagatatg gcggcgacta ctttgattat tggggccagg gcaccacact gaccgtgtct 360

agc 363

<210> 22

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> 19F3H2(hG1TM) amino acid sequence of heavy chain variable region

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Ala Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Glu Tyr Arg Tyr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 23

<211> 1353

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the heavy chain of 19F3H2(hG1TM)

<400> 23

caggtgcagc tggtgcagtc tggagcagag gtggtgaagc caggagcctc tgtgaaggtg 60

agctgtaagg ccagcggcta ctccttcacc ggctatacaa tgaactgggt gaggcaggca 120

ccaggacaga atctggagtg gatcggcctg atcaaccctt acaatgccgg cacctcttat 180

aaccagaagt ttcagggcaa ggtgaccctg acagtggaca agtccacctc tacagcctac 240

atggagctga gctccctgcg gagcgaggat acagccgtgt actattgcgc ccggtccgag 300

tacagatatg gcggcgacta ctttgattat tggggccagg gcaccacact gaccgtgtct 360

agcgcctcca caaaggggcc cagcgtgttt cctctcgccc cctcctccaa aagcaccagc 420

ggaggaaccg ctgctctcgg atgtctggtg aaggactact tccctgaacc cgtcaccgtg 480

agctggaata gcggcgctct gacaagcgga gtccatacat tccctgctgt gctgcaaagc 540

agcggactct attccctgtc cagcgtcgtc acagtgccca gcagcagcct gggcacccag 600

acctacatct gtaacgtcaa ccacaagccc tccaacacca aggtggacaa gaaagtggag 660

cccaaatcct gcgacaagac acacacctgt cccccctgtc ctgctcccga agctgctgga 720

gcccctagcg tcttcctctt tcctcccaaa cccaaggaca ccctcatgat cagcagaacc 780

cctgaagtca cctgtgtcgt cgtggatgtc agccatgagg accccgaggt gaaattcaac 840

tggtatgtcg atggcgtcga ggtgcacaac gccaaaacca agcccaggga ggaacagtac 900

aactccacct acagggtggt gtccgtgctg acagtcctcc accaggactg gctgaacggc 960

aaggagtaca agtgcaaggt gtccaacaag gctctccctg cccccattga gaagaccatc 1020

agcaaggcca aaggccaacc cagggagccc caggtctata cactgcctcc ctccagggac 1080

gaactcacca agaaccaggt gtccctgacc tgcctggtca agggctttta tcccagcgac 1140

atcgccgtcg agtgggagtc caacggacag cccgagaata actacaagac cacccctcct 1200

gtcctcgact ccgacggctc cttcttcctg tacagcaaac tgaccgtcga taaatctagg 1260

tggcagcagg gcaacgtgtt ctcttgttcc gtgatgcatg aagcactgca caaccattat 1320

acccagaagt ctctgagcct gtcccccggc aag 1353

<210> 24

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<223> 19F3H2(hG1TM) heavy chain amino acid sequence

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Ala Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Glu Tyr Arg Tyr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 25

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding the light chain variable region of 19F3L3

<400> 25

gacatcgtga tgacccagtc cccaagctcc ctggccgtgt ctgtgggaga gcgggtgaca 60

atctcctgta agtctagcca gtctctgctg aactcctcta atcagaagaa ctacctggcc 120

tggtatcagc agaagcccgg ccaggcccct aagctgctga tctacttcgc ctctaccagg 180

gagagcggag tgccagacag attctctggc agcggctccg gcacagactt caccctgaca 240

atcagctccc tgcaggcaga ggacgtggcc gtgtactatt gccagcagca ctacgatacc 300

ccctatacat ttggcggcgg caccaagctg gagatcaag 339

<210> 26

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of light chain variable region of 19F3L3

<400> 26

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Asp Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 27

<211> 660

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence encoding 19F3L3 light chain

<400> 27

gacatcgtga tgacccagtc cccaagctcc ctggccgtgt ctgtgggaga gcgggtgaca 60

atctcctgta agtctagcca gtctctgctg aactcctcta atcagaagaa ctacctggcc 120

tggtatcagc agaagcccgg ccaggcccct aagctgctga tctacttcgc ctctaccagg 180

gagagcggag tgccagacag attctctggc agcggctccg gcacagactt caccctgaca 240

atcagctccc tgcaggcaga ggacgtggcc gtgtactatt gccagcagca ctacgatacc 300

ccctatacat ttggcggcgg caccaagctg gagatcaagc gtacggtggc agccccatct 360

gtcttcattt ttccccctag tgacgagcag ctgaaatccg gaacagcctc tgtggtctgt 420

ctgctgaaca atttctaccc tcgcgaagcc aaggtgcagt ggaaagtcga taacgctctg 480

cagagtggca attcacagga gagcgtgact gaacaggact ccaaggattc tacctatagt 540

ctgagctcca ctctgaccct gtccaaagca gattacgaaa agcacaaagt gtatgcctgt 600

gaggtcaccc accaggggct gagttctcca gtcaccaaat ccttcaacag aggcgaatgt 660

<210> 28

<211> 220

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of 19F3L3 light chain

<400> 28

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Asp Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 29

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 30

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> 4G10H3V(M)

<400> 30

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Asn Ile Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg Leu Asp Tyr Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 31

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> 4G10L3V(M)

<400> 31

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Phe Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser

35 40 45

Leu Ile Gly Gly Thr Asn Asn Lys Ala Ser Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Arg

100 105 110

<210> 32

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 32

Gly Phe Ala Phe Ser Ser Tyr Asp

1 5

<210> 33

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 33

Ile Ser Gly Gly Gly Arg Tyr Thr

1 5

<210> 34

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 34

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr

1 5 10

<210> 35

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 35

Gln Asp Ile Asn Thr Tyr

1 5

<210> 36

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 36

Arg Ala Asn

1

<210> 37

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 37

Leu Gln Tyr Asp Glu Phe Pro Leu Thr

1 5

<210> 38

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 38

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 39

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 39

Ile Asn Pro Tyr Asn Asn Ile Thr

1 5

<210> 40

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 40

Ala Arg Leu Asp Tyr Arg Ser Tyr

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 41

Thr Gly Ala Val Thr Thr Ser Asn Phe

1 5

<210> 42

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 42

Gly Thr Asn

1

<210> 43

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 43

Ala Leu Trp Tyr Ser Asn His Trp Val

1 5

<210> 44

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 44

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 45

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 45

Ile Asn Pro Tyr Asn Ala Gly Thr

1 5

<210> 46

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 46

Ala Arg Ser Glu Tyr Arg Tyr Gly Gly Asp Tyr Phe Asp Tyr

1 5 10

<210> 47

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 47

Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr

1 5 10

<210> 48

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 48

Phe Ala Ser

1

<210> 49

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 49

Gln Gln His Tyr Asp Thr Pro Tyr Thr

1 5

<210> 50

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 50

Gly Gly Ser Ile Ser Asp Tyr Tyr

1 5

<210> 51

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 51

Ile Asn His Arg Gly Thr Thr

1 5

<210> 52

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 52

Ala Phe Gly Tyr Ser Asp Tyr Glu Tyr Asp Trp Phe Asp Pro

1 5 10

<210> 53

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 53

Gln Thr Ile Ser Ser Tyr

1 5

<210> 54

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 54

Asp Ala Ser

1

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 55

Gln Gln Arg Ser Asn Trp Pro Ile Thr

1 5

<210> 56

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence of heavy chain variable region H7v of H7L8

<400> 56

caggtgcagc tgcagcagtg gggagctgga ctgctgaaac ctagcgagac actgagcctg 60

acctgtgctg tgtacggcgg atctatcagc gattactact ggaactggat caggcagccc 120

cctggaaagg gactggaatg gatcggagag atcaaccaca ggggcaccac caactccaat 180

ccctctctga agagcagggt gacactgagc ctcgacacaa gcaagaatca gttcagcctg 240

aagctgaggt ccgtgaccgc tgctgataca gctgtgtact actgtgcctt cggctacagc 300

gattacgagt acgattggtt cgacccttgg ggccagggaa cactggttac agtgagctcc 360

<210> 57

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain variable region H7v of H7L8

<400> 57

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Ile Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Thr Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asp Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 58

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence of light chain variable region L8v of H7L8

<400> 58

gagatcgttc tgacccagag cccagctaca ctgagcctgt ctcctggaga gagggctaca 60

ctgtcctgca gagctagcca gaccatcagc agctacctgg cttggtacca gcagaagcct 120

ggccaagctc caaggctgct gatctacgac gcctctaata gggccaccgg catccctgct 180

agattctctg gaagcggcag cggaaccgac tttacactga caatcagctc cctggagccc 240

gaggatttcg ctgtttacta ctgtcagcag cgcagcaact ggcccatcac attcggacag 300

ggcacaaatc tggagatcaa g 321

<210> 59

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of light chain variable region L8v of H7L8

<400> 59

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 60

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Bs-PL022B (hG1TM) in immunoglobulin part of the heavy chain of the amino acid sequence

<400> 60

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Ile Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Thr Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asp Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 61

<211> 1350

<212> DNA

<213> Artificial Sequence

<220>

<223> Bs-PL022B (hG1TM) in immunoglobulin part of the heavy chain of nucleic acid sequences

<400> 61

caggtgcagc tgcagcagtg gggagctgga ctgctgaaac ctagcgagac actgagcctg 60

acctgtgctg tgtacggcgg atctatcagc gattactact ggaactggat caggcagccc 120

cctggaaagg gactggaatg gatcggagag atcaaccaca ggggcaccac caactccaat 180

ccctctctga agagcagggt gacactgagc ctcgacacaa gcaagaatca gttcagcctg 240

aagctgaggt ccgtgaccgc tgctgataca gctgtgtact actgtgcctt cggctacagc 300

gattacgagt acgattggtt cgacccttgg ggccagggaa cactggttac agtgagctcc 360

gcctccacca aggggcccag cgtgtttcct ctcgccccct cctccaaaag caccagcgga 420

ggaaccgctg ctctcggatg tctggtgaag gactacttcc ctgaacccgt caccgtgagc 480

tggaatagcg gcgctctgac aagcggagtc catacattcc ctgctgtgct gcaaagcagc 540

ggactctatt ccctgtccag cgtcgtcaca gtgcccagca gcagcctggg cacccagacc 600

tacatctgta acgtcaacca caagccctcc aacaccaagg tggacaagaa agtggagccc 660

aaatcctgcg acaagacaca cacctgtccc ccctgtcctg ctcccgaagc tgctggagcc 720

cctagcgtct tcctctttcc tcccaaaccc aaggacaccc tcatgatcag cagaacccct 780

gaagtcacct gtgtcgtcgt ggatgtcagc catgaggacc ccgaggtgaa attcaactgg 840

tatgtcgatg gcgtcgaggt gcacaacgcc aaaaccaagc ccagggagga acagtacaac 900

tccacctaca gggtggtgtc cgtgctgaca gtcctccacc aggactggct gaacggcaag 960

gagtacaagt gcaaggtgtc caacaaggct ctccctgccc ccattgagaa gaccatcagc 1020

aaggccaaag gccaacccag ggagccccag gtctatacac tgcctccctc cagggacgaa 1080

ctcaccaaga accaggtgtc cctgacctgc ctggtcaagg gcttttatcc cagcgacatc 1140

gccgtcgagt gggagtccaa cggacagccc gagaataact acaagaccac ccctcctgtc 1200

ctcgactccg acggctcctt cttcctgtac agcaaactga ccgtcgataa atctaggtgg 1260

cagcagggca acgtgttctc ttgttccgtg atgcatgaag cactgcacaa ccattatacc 1320

cagaagtctc tgagcctgtc ccccggcaag 1350

<210> 62

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Bs-PL022B (hG1TM) in immunoglobulin part of the light chain of amino acid sequence

<400> 62

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 63

<211> 642

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleic acid sequence of light chain of immunoglobulin fraction in Bs-PL022B (hG1TM)

<400> 63

gagatcgttc tgacccagag cccagctaca ctgagcctgt ctcctggaga gagggctaca 60

ctgtcctgca gagctagcca gaccatcagc agctacctgg cttggtacca gcagaagcct 120

ggccaagctc caaggctgct gatctacgac gcctctaata gggccaccgg catccctgct 180

agattctctg gaagcggcag cggaaccgac tttacactga caatcagctc cctggagccc 240

gaggatttcg ctgtttacta ctgtcagcag cgcagcaact ggcccatcac attcggacag 300

ggcacaaatc tggagatcaa gcgtacggtg gcagccccat ctgtcttcat ttttccccct 360

agtgacgagc agctgaaatc cggaacagcc tctgtggtct gtctgctgaa caatttctac 420

cctcgcgaag ccaaggtgca gtggaaagtc gataacgctc tgcagagtgg caattcacag 480

gagagcgtga ctgaacagga ctccaaggat tctacctata gtctgagctc cactctgacc 540

ctgtccaaag cagattacga aaagcacaaa gtgtatgcct gtgaggtcac ccaccagggg 600

ctgagttctc cagtcaccaa atccttcaac agaggcgaat gt 642

<210> 64

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> 14C12H1L1(M) light chain variable region 14C12L1(M) V

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

Claims

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

L234A and L235A;

L234A and G237A;

L235A and G237A;

or alternatively

L234A, L235A and G237A.

2. The method of claim 1, wherein the drug containing an immunoglobulin Fc fragment comprises an antibody and/or an Fc fusion protein;

3. The method of claim 2, wherein the antibody is an immune checkpoint inhibitor.

4. The method of any one of claims 1-3, wherein the antibody is a bispecific antibody or a multispecific antibody.

5. The method of any one of claims 1-4, wherein the antibody targets:

6. The method of any one of claims 4 to 5, wherein the bispecific antibody targets PD-1 and CTLA4 comprising:

targeting a first protein functional region of PD-1, and

targeting a second protein domain of CTLA 4;

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: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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:38-40, respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:41-43, respectively;

alternatively, the first and second electrodes may be,

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

the immunoglobulin is human IgG; and is

7. The method of claim 6, 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 variable region of the light chain 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 14, SEQ ID NO 18 and SEQ ID NO 30; and the amino acid sequence of the variable region of the light chain 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;

alternatively, the first and second electrodes may be,

the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 30; and the amino acid sequence of the variable region of the light chain 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 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 SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64.

8. The method of claim 6 or 7, wherein the bispecific antibody is selected from any one of (1) - (18) as follows:

(1)

(2)

(3)

(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)

(7)

(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)

(10)

(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)

(13)

(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)

(16)

(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)

9. The method of any one of claims 4 to 5, wherein the bispecific antibody targets CD73 and PD-1, comprising:

targeting a first protein functional region of CD73, and

a second protein functional region targeted to 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: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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:32-34 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:35-37 respectively;

alternatively, the first and second electrodes may be,

the first protein functional region is a single-chain antibody, and the second protein functional region is 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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:44-46 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:47-49 respectively;

the immunoglobulin is human IgG; and is

10. The method of claim 9, 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 variable region of the light chain 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 SEQ ID NO 2 and SEQ ID NO 6; and the amino acid sequence of the variable region of the light chain of the single-chain antibody is selected from SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64;

alternatively, the first and second liquid crystal display panels may be,

11. The method of claim 9 or 10, wherein the bispecific antibody is selected from any one of (1) - (6) as follows:

(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)

(3)

(4)

(5)

(6)

12. The method of claim 4 or 5, wherein the bispecific antibody targets LAG3 and PD-1, comprising:

a first protein functional region targeting LAG3, and

a second protein functional region targeted to 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 single-chain antibody, wherein the heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NOs:32-34 respectively, and the light chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown as SEQ ID NOs:35-37 respectively;

alternatively, the first and second electrodes may be,

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

the immunoglobulin is human IgG; and is

13. The method of claim 12, 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 variable region of the light chain 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 SEQ ID NO 2 and SEQ ID NO 6; and the amino acid sequence of the variable region of the light chain of the single-chain antibody is selected from SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 64;

alternatively, the first and second electrodes may be,

14. The method of claim 12 or 13, wherein the bispecific antibody is selected from any one of (1) - (6) as follows:

(1)

(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)

(4)

(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)

15. The method of any one of claims 6 to 14, wherein the immunoglobulin heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, and the immunoglobulin light chain constant region is selected from the light chain constant regions of human IgG1, IgG2, IgG3, or IgG 4;

preferably, the immunoglobulin heavy chain constant region is human Ig gamma-1chain Cregion or human Ig gamma-4chain C region, and the immunoglobulin light chain constant region is human Ig kappa chain C region.

16. The method of any one of claims 1 to 15, wherein the immune cell is a human immune cell, such as a human macrophage.

17. The method of any one of claims 1 to 16, wherein the method is a method of non-therapeutic purpose.

18. The method of any one of claims 1 to 17, wherein the method is a method for pharmaceutical purposes.

19. A method of increasing the efficacy and/or safety of a drug comprising an Fc fragment of an immunoglobulin, wherein the level of IL-8 and/or IL-6 secreted by an immune cell mediated by a drug comprising an Fc fragment of an immunoglobulin is reduced by the method of any one of claims 1 to 18.