WO2022188867A1

WO2022188867A1 - Method for improving safety of drug containing immunoglobulin fc fragment

Info

Publication number: WO2022188867A1
Application number: PCT/CN2022/080392
Authority: WO
Inventors: 张鹏; 李百勇; 夏瑜; 王忠民
Original assignee: 中山康方生物医药有限公司
Priority date: 2021-03-12
Filing date: 2022-03-11
Publication date: 2022-09-15
Also published as: CN115073588B; CN118530341A; CN115073588A; CN118530342A

Abstract

Provided is a method for optimizing a drug containing an immunoglobulin Fc fragment to improve the safety and/or efficacy of the drug. Specifically, provided is a method for reducing or blocking the level of IL-8 and/or IL-6 secreted by an immune cell mediated by a drug containing an immunoglobulin Fc fragment. The method can effectively improve the safety and/or efficacy of the drug containing the immunoglobulin Fc fragment.

Description

Methods for improving the safety of drugs containing immunoglobulin Fc fragments

technical field

The present invention belongs to the field of tumor therapy and molecular immunology, and relates to a method for optimizing the safety and/or effectiveness of a drug (such as an antibody or Fc fusion protein) containing an immunoglobulin Fc fragment. In particular, the present invention relates to a method of reducing or blocking the levels of IL-8 and/or IL-6 secreted by immune cells mediated by drugs containing immunoglobulin Fc fragments (eg, antibodies or Fc fusion proteins).

Background technique

Fc receptors are immunoglobulin family proteins that are expressed on the surface of specific immune cells or somatic cells and are used to recognize the Fc region of antibodies to mediate immune responses. After the Fab region of the antibody recognizes the antigen, the Fc region of the antibody binds to Fc receptors on immune cells (such as killer cells) to activate effector cells.

According to the different types of antibodies recognized by Fc receptors and the different expressing cells, Fc receptors are mainly divided into three types: FcγR, FcαR and FcεR. FcγR can be further divided into FcγRI (also known as CD64), FcγRII (also known as CD32), FcγRIII (also known as CD16) and FcRn (also known as Neonatal Fc receptor) four subtypes. Among them, FcγRI, FcγRII and FcγRIII are closely related to ADCC effect. FcγRIII is the most important molecule mediating ADCC. It has two isoforms, FcγRIIIa and FcγRIIIb, which are highly homologous in different cell types. There is a high-affinity FcγRIIIa subtype caused by mononuclear stem polymorphism (SNP) in the FcγRIIIa population. The two subtypes are called FcγRIIIa_V158 and low-affinity FcγRIIIa_F158, respectively. FcγRI has a high affinity for the Fc region of IgG and participates in the ADCC process; FcγRII has three subtypes: FcγRIIa, FcγRIIb and FcγRIIc (also known as CD32a, CD32b, CD32c), of which FcγRIIa has ADCC activity; FcγRIIa exists in the population The two isoforms due to single nucleotide mutations are called FcγRIIa_H131 and FcγRIIa_R131 (Hogarth PM, Pietersz GA. 2012, Nature Review Drug Discovery, 11(4):311-331).

The IgG family consists of four members, IgG1, IgG2, IgG3 and IgG4, which have amino acid differences in the fragment crystallizable (Fc) region of their heavy chain constant regions, resulting in their different affinities with FcγRs. IgG1 is the most subtype in the human body and the most used subtype in monoclonal antibody drugs. IgG1 can bind to various FcγRs. IgG2 has the weakest affinity for FcγRs, but IgG2 is still able to bind to FcγRIIa. IgG3 has the strongest binding ability to FcγRs. IgG4 molecules bind weakly to FcγRs other than FcγRI. The IgG4 antibody subtype is unstable and prone to breakage in the hinge region, which leads to Fab-arm exchange, forming half-molecular and bispecific functional monovalent antibodies (Aalberse R.C.et al.Clin.Exp.Allergy.2009;39(4):469 -77.); The introduction of S228P mutation into the hinge region of IgG4 antibody heavy chain can stabilize IgG4 molecule and prevent the formation of half-molecule (Shirley J Peters et al. J Biol Chem. 2012 13; 287(29): 24525-33.).

ADCR (antibody-dependent cytokine release) refers to antibody-dependent cytokine release. The Fab segment of the antibody binds to the antigenic epitope of the tumor cell, and the Fc segment cross-binding with the Fc receptor (Fc Receptor, FcR) on the effector cell surface, through crosslinking Activation of effector cells, resulting in a large amount of cytokines secreted by activated effector cells, such as IL (interleukin)-1, IL-6, IL-8, IL-10, MCP (monocyte chemotactic protein)-1, etc. , of which IL-6 is the main inflammatory mediator. These cytokines will weaken the efficacy of immunotherapy and increase immune-related adverse reactions, which may lead to multiple organ failure and death in severe cases.

Interleukin-8 (interleutin-8, IL-8) is a chemotactic cytokine (Chemotactic cytokines), belonging to the CXC-α subfamily (also known as CXCL-8). In normal humans, it is mainly secreted by monocytes, immune cells, epithelial cells, etc., and is involved in inflammation and immune defense responses in the body; its receptor (CXCR) is a dimer sugar composed of two subunits of 59 and 67 kDa. The protein, belonging to the G protein-coupled receptor superfamily, has two subtypes, CXCR1 and CXCR2. IL-8 plays an important role in the proliferation of normal cells and tumor cells, especially in promoting the occurrence and development of tumors. Studies have shown that IL-8 can promote tumorigenesis; tumor cells themselves can also secrete IL-8 to promote tumor growth and metastasis (Lo MC et al. Cancer letters, 2013, 335(1):81-92.). Therefore, IL-8 has become an indispensable and important inflammatory factor in the tumor microenvironment.

As a pro-inflammatory factor, IL-8 is closely related to the occurrence and development of tumors. In the process of methylarsonate-induced malignant transformation of non-renal cancer cells, the expression of IL-8 gene is increased, and IL-8 gene silencing can significantly inhibit the growth of transplanted tumors in mice. In addition, the level of IL-8 decreases. It 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 tumor growth and metastasis (Escudero -Lourdes C et al. Toxicology and applied pharmacology, 2012, 258(1):10-18.). Inoue et al. found that IL-8 induced the malignant transformation and increased invasiveness of non-neoplastic bladder cell line (233JP), while the probability of malignant transformation of 233JP cells was significantly reduced in IL-8 knockout mice (Inoue K et al. al. Cancer Res, 2000, 60(8):2290-2299.). In addition, in prostate cancer, IL-8 can promote the occurrence of castration-resistant prostate cancer (CRPC) in patients (Chen K et al. Cancer research, 2015, 75(10): 1992-2004.), and is closely related to tumor therapy (Araki S et al. Cancer Res, 2007, 67(14): 6854-6862.); gene silencing of IL-8 or its receptors can induce cell cycle arrest in tumor cells and inhibit tumor proliferation ( Singh RK, Lokeshwar BL. Molecul Cancer, 2009, 8:57.). The above studies show that the level of IL-8 is closely related to the occurrence and development of tumors. Further research (Mian BM et al. Clin Cancer Res, 2003, 9(8): 3167-3175.) showed that IL-8 can be used as a new target for tumor therapy. In the tumor model of bladder cancer, the use of anti-IL-8 antibody can significantly inhibit tumor growth.

IL-6 is rapidly produced mainly by macrophages in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and protects damaged tissues by removing infectious agents, inducing acute phase and immune responses effect.

IL-6 is also responsive to ligands including Toll-like receptor (TLR) and pro-inflammatory cytokines such as IL-1 and TNF-[alpha]. In infected lesions, IL-6 is produced by stimulation of TLRs on monocytes and macrophages, each of which recognizes the corresponding bacterial, viral or fungal components such as lipopolysaccharide, CpG DNA, double- or single-stranded RNA And peptidoglycan, PAMP. IL-6 can also be produced in non-infectious inflammations such as burns and wounds, and its levels depend on the severity of the disease. Damaged or necrotic cells and damaged or degraded extracellular matrix are released through DAMP mode such as mitochondrial DNA, high mobility group box chromosomal protein 1 (HMGB1), heat shock protein and S100 molecules, etc., and then stimulate the corresponding TLR production including IL-6 of proinflammatory cytokines.

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

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 achieved great success in tumor immunotherapy, but they have also led to a completely new toxicity spectrum due to off-target effects. Serious immune-related adverse events (irAEs), especially in major organs, including heart, lung, and brain, can be life-threatening (Bergqvist V, et al. Cancer Immunol Immunother. 2017;66(5):581-592.;Gomatou G et al.Respiration.2020;1:1-11.;Joshi MN et al.Clin Endocrinol(Oxf).2016;85(3):331-9.;Prieux-Klotz C et al.Target Oncol.2017;12 (3): 301-308.; Tajiri K et al. Jpn J Clin Oncol. 2018; 48(1): 7-12.). Existing data indicate that ICI may induce off-target effects through four mechanisms, including direct binding to immune checkpoint molecules expressed on the surface of normal cells, activation of complement hypersensitivity; the existence of homologous antigens/epitopes in normal tissues and tumor cells; production of autoantibodies; Increased levels of pro-inflammatory cytokines, such as IL-6, etc. (Martins F et al., The Lancet Oncology, 20(1), e54–e64.).

Current anti-IL-6 therapies, such as tocilizumab, a recombinant humanized anti-IL-6R monoclonal antibody, have been used to treat acute-phase severe irAEs, severe or refractory arthritis, large vessel vasculitis, Uveitis, myocarditis, pneumonia, myasthenia gravis, etc. (Martins F et al., The Lancet Oncology, 20(1), e54–e64.).

Fc fusion protein drugs, such as IL-2-Fc fusion protein, have been proven to be used to treat tumors, but due to their inherent toxicity, such as IL-2-Fc fusion protein can cause lethal capillary Vascular leak disease, and induce proliferation of immunosuppressive Treg cells, affecting its anti-tumor activity, if its Fc fragment can further induce immune cells to secrete IL-8 and/or IL-6, it will significantly affect its anti-tumor effectiveness and safety. These all limit its clinical application.

To sum up, for immune checkpoint inhibitors, Fc fusion protein drugs, especially antibody drugs targeting immune checkpoints and Fc fusion protein drugs with cytokines, chemokines and their ligands as the mechanism of action, inhibit Its effect of inducing immune cells to secrete IL-8 and/or IL-6 is of great significance for improving the efficacy and/or safety of the drug.

SUMMARY OF THE INVENTION

After in-depth research and creative work, the inventors have developed anti-dual immune checkpoint inhibitors (anti-PD-1/CTLA4 bispecific antibody, anti-PD-1/CD73 bispecific antibody, anti-PD-1/LAG3 bispecific antibody Corresponding transformation of the Fc end of an Fc-based antibody, etc., or an Fc fusion protein with immunomodulatory biological activity, such as a fusion protein of IL-2 and Fc, can effectively reduce or eliminate immune checkpoint therapy antibodies or fusion protein-mediated or Induced unintended activity of immune cells to secrete IL-6 and/or IL-8, thereby increasing the safety and/or efficacy of immune checkpoint inhibitors and fusion protein drugs. The following invention is thus provided:

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

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

L234A and L235A;

L234A and G237A;

L235A and G237A;

or

L234A, L235A and G237A.

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

Optionally, the medicine containing the immunoglobulin Fc fragment further comprises one or more pharmaceutically acceptable excipients.

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

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

In some embodiments of the present invention, the method, wherein the drug containing an immunoglobulin Fc fragment is an antibody and an Fc fusion protein.

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

In some embodiments of the present invention, the method, wherein the immunoglobulin Fc fragment-containing drug is composed of an antibody and/or Fc fusion protein as an active ingredient (API), and one or more pharmaceutically Acceptable excipient composition.

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

In some embodiments of the present invention, the method, wherein the immunoglobulin Fc fragment-containing drug is composed of an antibody and/or Fc fusion protein as the sole active ingredient (API), and one or more pharmaceutical agents composition of acceptable excipients.

Those skilled in the art can understand that when the drug containing the immunoglobulin Fc fragment contains one or more pharmaceutically acceptable excipients, it is actually a pharmaceutical composition. Various dosage forms, such as injections, can be prepared according to the skills of those skilled in the art.

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

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

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

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

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

targeting the first protein domain of PD-1, and

Targets the second protein functional region of CTLA4;

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

in,

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

or,

Described immunoglobulin, its heavy chain variable region comprises the HCDR1-HCDR3 shown in amino acid sequence respectively as SEQ ID NOs:38-40, and its light chain variable region comprises aminoacid sequence respectively as SEQ ID NOs:41-43 Shown LCDR1-LCDR3; And described single chain antibody, its heavy chain variable region comprises the HCDR1-HCDR3 shown in SEQ ID NOs:32-34 respectively, and its light chain variable region comprises amino acid sequence respectively. LCDR1-LCDR3 as shown in SEQ ID NOs: 35-37;

in,

The immunoglobulin is human IgG;

There are two single-chain antibodies, and one end of each single-chain antibody is respectively connected to the C-terminus of the two heavy chains of immunoglobulin.

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

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from 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 SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31;

or,

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: 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 the group consisting of SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO:64.

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

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

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

(7)

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

(8)

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

(9)

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

(10)

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

(11)

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

(12)

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

(13)

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

(14)

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

(15)

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

(16)

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

(17)

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

(18)

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

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

targeting the first protein domain of CD73, and

Targeting the second protein functional region of PD-1;

in,

Described immunoglobulin, its heavy chain variable region comprises the HCDR1-HCDR3 shown in amino acid sequence as SEQ ID NOs:44-46 respectively, and its light chain variable region comprises aminoacid sequence respectively as SEQ ID NOs:47-49 Shown LCDR1-LCDR3; And described single chain antibody, its heavy chain variable region comprises the HCDR1-HCDR3 shown in SEQ ID NOs:32-34 respectively, and its light chain variable region comprises amino acid sequence respectively. LCDR1-LCDR3 as shown in SEQ ID NOs: 35-37;

or,

Described immunoglobulin, its heavy chain variable region comprises the HCDR1-HCDR3 shown in amino acid sequence respectively as SEQ ID NOs:32-34, and its light chain variable region comprises aminoacid sequence respectively as SEQ ID NOs:35-37 Shown LCDR1-LCDR3; And described single chain antibody, its heavy chain variable region comprises the HCDR1-HCDR3 shown in SEQ ID NOs:44-46 respectively, and its light chain variable region comprises amino acid sequence respectively. LCDR1-LCDR3 as shown in SEQ ID NOs: 47-49;

in,

The immunoglobulin is human IgG;

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

The amino acid sequence of the variable region of the heavy chain 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 SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region of the 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: 6; NO:8 and SEQ ID NO:64;

or,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from 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 light chain variable region of the single chain antibody The amino acid sequence is selected from SEQ ID NO:26.

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

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

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

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

targeting the first protein domain of LAG3, and

Targeting the second protein functional region of PD-1;

in,

Described immunoglobulin, its heavy chain variable region comprises the HCDR1-HCDR3 shown in amino acid sequence as SEQ ID NOs:50-52 respectively, and its light chain variable region comprises aminoacid sequence respectively as SEQ ID NOs:53-55 Shown LCDR1-LCDR3; And described single chain antibody, its heavy chain variable region comprises the HCDR1-HCDR3 shown in SEQ ID NOs:32-34 respectively, and its light chain variable region comprises amino acid sequence respectively. LCDR1-LCDR3 as shown in SEQ ID NOs: 35-37;

or,

Described immunoglobulin, its heavy chain variable region comprises the HCDR1-HCDR3 shown in amino acid sequence respectively as SEQ ID NOs:32-34, and its light chain variable region comprises aminoacid sequence respectively as SEQ ID NOs:35-37 Shown LCDR1-LCDR3; And described single chain antibody, its heavy chain variable region comprises the HCDR1-HCDR3 shown in SEQ ID NOs:50-52 respectively, and its light chain variable region comprises amino acid sequence respectively. LCDR1-LCDR3 as shown in SEQ ID NOs: 53-55;

in,

The immunoglobulin is human IgG;

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

The amino acid sequence of the variable region of the heavy chain 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 SEQ ID NO: 59; The amino acid sequence of the heavy chain variable region of the 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: 6; NO:8 and SEQ ID NO:64;

or,

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

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

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

In some embodiments of the invention, the method, wherein the heavy chain constant region of the immunoglobulin of the bispecific antibody is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the The light chain constant region of the immunoglobulin of the bispecific antibody is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4;

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

When the antibody adopts the above-mentioned heavy chain constant region and light chain constant region, the immunoglobulin Fc fragment of the bispecific antibody contains the aforementioned mutation, that is, the heavy chain of the immunoglobulin of the bispecific antibody The constant region contains the aforementioned mutations. E.g:

In some embodiments of the invention, the method, wherein the heavy chain constant region of the immunoglobulin of the bispecific antibody is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the The light chain constant region of the immunoglobulin of the bispecific antibody is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4; and the heavy chain constant region of the immunoglobulin of the bispecific antibody according to the EU numbering system Contains the following mutations:

L234A and L235A;

L234A and G237A;

L235A and G237A;

or

L234A, L235A and G237A.

Another example:

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

L234A and L235A;

L234A and G237A;

L235A and G237A;

or

L234A, L235A and G237A.

In some embodiments of the present invention, the method, wherein the immune cells are human immune cells, such as human macrophages.

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

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

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

Another aspect of the present invention relates to a method for improving the efficacy and/or safety of a drug containing an immunoglobulin Fc fragment, wherein, by the method of any one of the present invention, the reduction of immunoglobulin Fc containing Fragments of drug-mediated or induced levels of IL-8 and/or IL-6 secreted by immune cells. In some embodiments of the present invention, the method, wherein the method is a pharmaceutical method.

Those skilled in the art know that the variable regions of light and heavy chains determine antigen binding; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) Comprising HCDR1, HCDR2, HCDR3, the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; named by Kabat et al, see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vols 1-3, NIH Publication 91-3242, Bethesda Md).

The amino acid sequence of the CDR region of the antibody sequence involved in the present invention is analyzed by technical means well-known to those skilled in the art, for example, through the VBASE2 database, and the results are as follows:

(1) 14C12

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

The amino acid sequences of the three CDR regions in the variable region of its heavy chain 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 three 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 in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

The amino acid sequence of the three CDR regions of the variable region of its heavy chain is the same as that of 14C12.

The amino acid sequence of the three CDR regions of the light chain variable region is the same as that of 14C12.

(3)4G10

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

HCDR1: GYSFTGYT (SEQ ID NO: 38)

HCDR2: INPYNNIT (SEQ ID NO: 39)

HCDR3:ARLDYRSY (SEQ ID NO:40)

LCDR1:TGAVTSNF (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 in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20;

The amino acid sequence of the three CDR regions of the variable region of the heavy chain is the same as that of 4G10.

The amino acid sequence of the three CDR regions of the light chain variable region is the same as that of 4G10.

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

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

(6)19F3H2(hG1TM)

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in 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 in SEQ ID NO:57, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:59;

HCDR1: GGSISDYY (SEQ ID NO: 50)

HCDR2: INHRGTT (SEQ ID NO: 51)

HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 52)

LCDR1: QTISSY (SEQ ID NO: 53)

LCDR2: DAS (SEQ ID NO: 54)

LCDR3: QQRSNWPIT (SEQ ID NO: 55)

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, when referring to the amino acid sequence of PD-1 protein (Programmed cell death protein 1), it includes but is not limited to the full length of PD-1 protein (NCBI GenBank: NP_005009.2), or PD- 1's extracellular fragment PD-1ECD or a fragment comprising PD-1ECD; also includes a fusion protein of PD-1ECD, such as a fragment fused to a mouse or human IgG Fc protein fragment (mFc or hFc). However, those skilled in the art understand that in the amino acid sequence of PD-1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" shall include all such sequences, as well as natural or artificial variants thereof. And, when describing the sequence fragment of PD-1 protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.

As used herein, when referring to the amino acid sequence of CTLA4 protein, it includes, but is not limited to, the full length of CTLA4 protein (NCBI Genebank ID: NP_054862.1), or the extracellular fragment CTLA4 ECD or a fragment comprising CTLA4 ECD ; also includes fusion proteins of CTLA4 ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of CTLA4 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "CTLA4 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the CTLA4 protein, it includes the CTLA4 sequence fragment, and also includes the corresponding sequence fragment in its natural or artificial variants.

As used herein, when referring to the amino acid sequence of CD73 protein, it includes, but is not limited to, the full length of CD73 protein (NCBI Genebank ID: NP_054862.1), or the extracellular fragment CD73 ECD or CD73 ECD-containing Fragments; also include fusion proteins of CD73 ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of CD73 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "CD73 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when a sequence fragment of the CD73 protein is described, the CD73 sequence fragment is included, as well as the corresponding sequence fragment in its natural or artificial variants.

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 of LAG3, the LAG3 ECD, or the LAG3 ECD-containing Fragments; also include fusion proteins of LAG3 ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced in the amino acid sequence of LAG3 protein without affecting its biological function. Therefore, in the present invention, the term "LAG3 protein" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the LAG3 protein, it includes the LAG3 sequence fragment, and also includes the corresponding sequence fragment in natural or artificial variants thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule generally 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 mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 CH3). 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 regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair, respectively, form the antibody binding site. The assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia (1989) Definition of 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. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies are typically obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, eg, U.S. Patent 4,816,567).

As used herein, the term "humanized antibody" refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody) The antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve 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., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).

As used herein, the term "isolated" or "isolated" refers to artificially obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of. The terms "isolated" or "isolated" do not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as λ phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

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

As used herein, the term " _KD " 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, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies exhibit a dissociation equilibrium constant (K _D ) of less than about ^10-5 M, eg, less than about ^10-6 M, ^10-7 M, ^10-8 M, ^10-9 M, or ^10-10 M or less Binds antigen (eg, PD-1 protein). KD can be determined using methods known to those skilled in the art, eg, using a _Fortebio Molecular Interactometer.

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. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent. 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, the desired effect. For example, a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor); a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications. Determination of such effective amounts is well within the purview of those skilled in the art. 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 patient's general condition such as age, weight and sex, the mode of administration of the drug, and other concurrently administered treatments and many more.

As used herein, the term "complete elimination" means no or very low binding signal detected by existing instrumentation (eg, Fortebio Octet Molecular Interactometer). In one embodiment of the invention, very low binding signal means that the binding signal is below 0.1 nm.

In the present invention, the term "fusion protein (FP)" refers to purposefully linking two or more genes encoding functional proteins together to express the desired protein. A protein product obtained after the coding regions of or multiple genes are connected end to end, and the genes are controlled by the same regulatory sequence.

In the present invention, the term "immunoglobulin (Ig) fusion protein" refers to a recombinant protein with the above two partial domains that is expressed in eukaryotic or prokaryotic cells by linking a target gene with an Ig partial fragment gene at the gene level . According to the connection between the target protein and different fragments of Ig, it can be divided into two categories: one is Fab (Fv) fusion protein; the other is Fc fusion protein.

In the present invention, the term "Fc fusion protein" refers to a novel protein produced by fusing a certain biologically active functional protein molecule with an Fc fragment using techniques such as genetic engineering. or ligand) soluble ligand (or receptor) molecules or other active substances (such as cytokines) that need to prolong the half-life. Fc fusion proteins mainly combine biologically active proteins with the hinge region and the CH2 and CH3 regions of Ig.

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

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

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

In the present invention, unless otherwise specified, the letters before the site represent the amino acid before mutation, and the letters after the site represent the amino acid after mutation.

Beneficial Effects of Invention

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

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

(2) The present invention can also effectively eliminate the unexpected secretion of IL-6 and/or IL-8 in medicines containing Fc fragments, such as Fc fusion protein medicines.

Description of drawings

Figure 1: In the co-culture system of CHO-K1-PD1-CTLA4 cells and human macrophages, amino acid mutations in the Fc segment effectively abolished the secretion of IL-8 from human macrophages mediated by anti-PD-1/CTLA4 bispecific antibody .

Figure 2: In the co-culture system of CHO-K1-PD1-CTLA4 cells and human macrophages, amino acid mutations in the Fc segment effectively abolished the secretion of IL-6 from human macrophages mediated by anti-PD-1/CTLA4 bispecific antibody .

Figure 3: In the co-culture system of CHO-K1-PD1 cells and human macrophages, amino acid mutations in the Fc segment effectively abolished the secretion of IL-8 from human macrophages mediated by anti-PD-1/CD73 bispecific antibody.

Figure 4: In the co-culture system of CHO-K1-PD1 cells and human macrophages, amino acid mutations in the Fc segment effectively abolished the secretion of IL-6 from human macrophages mediated by anti-PD-1/CD73 bispecific antibody.

Figure 5: In the co-culture system of U87-MG cells and human macrophages, amino acid mutations in the Fc segment effectively abolished the secretion of IL-8 from human macrophages mediated by anti-PD-1/CD73 bispecific antibody.

Figure 6: In the co-culture system of U87-MG cells and human macrophages, the amino acid mutation of the Fc segment effectively eliminated the secretion of IL-6 by human macrophages mediated by anti-PD-1/CD73 bispecific antibody.

Figure 7: In the co-culture system of CHO-K1-PD1-LAG3 cells and human macrophages, the amino acid mutation in the Fc segment effectively eliminated the secretion of IL-8 from human macrophages mediated by anti-PD-1/LAG3 bispecific antibody .

Figure 8: In the co-culture system of CHO-K1-PD1-LAG3 cells and human macrophages, the amino acid mutation in the Fc segment effectively eliminated the secretion of IL-6 by human macrophages mediated by anti-PD-1/LAG3 bispecific antibody .

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. Those who do not indicate specific technology or conditions in the embodiment, according to the technology or conditions described in the literature in this area (for example, with reference to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang et al., 3rd edition, Science Press) or follow the product instructions. If the reagents or instruments used do not indicate the manufacturer, they are conventional products that can be purchased in the market.

In the following experimental examples of the present invention, the BALB/c mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

In the experimental example, the IgG4 subtype anti-PD-1 antibody Nivolumab (trade name Opdivo) carrying the S228P mutation (Wang C et al. Cancer Immunol Res. 2014; 2(9): 846-56.) was used, and the Fc end was retained. The IgG1 subtype Ipilimumab (trade name Yervoy) with FcγR function was purchased from Bristol-Myers Squibb as the control antibody; the IgG4 subtype Relatlimab was used as the control antibody, which was made by Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20200630.

In the following experimental examples of the present invention, the heavy chain variable region and light chain variable region sequences of the anti-CD73 antibody 19F3H2L3 (hG1WT) used are the same as those of 19F3H2L3 (hG1TM) in Preparation Example 2, and the constant region fragment adopts Ig gamma -1 chain C region, ACCESSION:P01857 is the heavy chain constant region, Ig kappa chain C region, ACCESSION:P01834 is the light chain constant region.

In the following experimental examples of the present invention, the used isotype control antibodies, namely hIgG1 and hIgG4, are antibodies targeting human anti-egg lysosome (HEL), and the variable region sequences of these antibodies are from the published by Acierno et al. Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.), the constant region fragment of hIgG1 adopts Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region, Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region; hIgG4 heavy chain constant region adopts the Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region And introduce S228P mutation to improve stability, Ig kappa chain C region, ACCESSION: P01834 is the light chain constant region; hIgG1, hIgG1 (DM) and hIgG4 are all made in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

Preparation Example 1: Design and Preparation of Anti-PD-1/CTLA4 Bispecific Antibody BiAb004 (hG1TM)

The structural pattern of the bispecific antibody BiAb004 (hG1TM) belongs to the Morrison pattern (IgG-scFv), that is, at the C-terminus of both heavy chains of an immunoglobulin part (IgG) antibody, the scFv fragments of the other antibody are connected by linker fragments. , its immunoglobulin part is based on PD-1 antibody, the scFv fragment is based on anti-CTLA4 antibody, and the middle is linked by a linker fragment.

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

Anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 (hG1WT) have heavy chain and light chain variable region amino acid sequences and coding nucleic acid sequences that are identical to 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A, respectively.

(1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

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

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

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

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

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

Nucleic acid sequence encoding 14C12H1L1 (hG1WT) heavy chain variable region (14C12H1V): (354bp)

Amino acid sequence of 14C12H1L1 (hG1WT) heavy chain variable region (14C12H1V): (118aa)

Nucleic acid sequence encoding 14C12H1L1 (hG1WT) light chain variable region (14C12L1V): (321bp)

Amino acid sequence of 14C12H1L1 (hG1WT) light chain variable region (14C12L1V): (107aa)

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

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

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

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

2. Sequence Design of Anti-CTLA4 Antibody

The amino acid sequences of the heavy chain and light chain of the anti-CTLA4 antibody 4G10 and its humanized antibody 4G10H3L3, and the coding nucleic acid sequences are respectively identical to 4G10 and 4G10H3L3 in Chinese Patent Publication CN 106967172A.

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

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

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

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

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

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

Nucleic acid sequence encoding 4G10H3L3 heavy chain variable region (4G10H3V): (345bp)

Amino acid sequence of 4G10H3L3 heavy chain variable region (4G10H3V): (115aa)

Nucleic acid sequence encoding 4G10H3L3 light chain variable region (4G10L3V): (327bp)

Amino acid sequence of 4G10H3L3 light chain variable region (4G10L3V): (109aa)

3. Sequence design of bispecific antibody BiAb004(M)

The structural pattern of the bispecific antibody BiAb004(M) belongs to the Morrison pattern (IgG-scFv), that is, the C-terminus of the two heavy chains of one IgG antibody is connected to the scFv fragment of the other antibody through a linker fragment. The design composition of the chain is shown in Table 1 below.

Table 1: Sequence design of BiAb004(M)

From Table 1 above:

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

In addition, 4G10H3V (M) and 4G10L3V (M) in the scFv fragment of BiAb004 (M) antibody in the above table 1 are based on 4G10H3V and 4G10L3V. Individual amino acids in the framework region were mutated, which effectively optimized the antibody structure, increasing its effectiveness.

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

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

In order to distinguish it from the mutated antibody BiAb004(hG1TM) hereinafter, BiAb004(M) is regarded as "wild type", also referred to as BiAb004(hG1WT) in the examples of the present invention. BiAb004 (hG1WT) adopts the Ig gamma-1 chain C region, ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin part, and the Ig kappa chain C region, ACCESSION:P01834 as the light chain constant region of the immunoglobulin part.

4. Non-variable region amino acid mutation design based on humanized bispecific antibody BiAb004 (hG1WT)

On the basis of BiAb004 (hG1WT) obtained above, the present inventors introduced a point mutation (L234A) from leucine to alanine at the 234th position of its heavy chain according to the EU numbering system, the 235th A leucine-to-alanine point mutation (L235A) was introduced at the No. 237 position, and a glycine-to-alanine point mutation (G237A) was introduced at the No. 237 position to obtain BiAb004 (hG1TM). The rest of the amino acid sequence is exactly the same as BiAb004 (hG1WT).

Preparation Example 2: Design and Preparation of Anti-PD-1/CD73 Bispecific Antibody NTPDV2 (hG1TM)

The structural pattern of the bispecific antibody NTPDV2 (hG1TM) belongs to the Morrison pattern (IgG-scFv), that is, the C-terminus of the two heavy chains of one IgG antibody is connected to the scFv fragment of the other antibody through a linker fragment. The design composition of the chain is shown in Table 2 below. NTPDV2 (hG1TM) uses the Ig gamma-1 chain C region, ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin part, and uses the Ig kappa chain C region, ACCESSION:P01834 as the light chain constant region of the immunoglobulin part, and On this basis, three mutations were made according to the EU numbering system: L234A, L235A and G237A.

Table 2: Sequence design of NTPDV2 (hG1TM)

From Table 2 above:

The amino acid sequence of Linker is shown in the preceding SEQ ID NO:29.

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

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

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

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

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

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

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

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

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

19F3L3 is used as the light chain of the immunoglobulin part of NTPDV2 (hG1TM), and its amino acid sequence is as follows (220aa), wherein the CDR region is underlined and shown in bold:

Preparation Example 3: Design and Preparation of Anti-PD-1/LAG3 Bispecific Antibody Bs-PL022B (hG1TM)

The structural pattern of the bispecific antibody Bs-PL022B (hG1TM) belongs to the Morrison pattern (IgG-scFv), that is, at the C-terminus of the two heavy chains of one IgG antibody, the scFv fragments of the other antibody are connected by linking fragments, and its heavy chain and the design composition of the light chain is shown in Table 3 below. Bs-PL022B (hG1TM) uses the Ig gamma-1 chain C region, ACCESSION:P01857 as the heavy chain constant region of the immunoglobulin part, and uses the Ig kappa chain C region, ACCESSION:P01834 as the light chain constant region of the immunoglobulin part , and on this basis, three mutations were made according to the EU numbering system: L234A, L235A, L237A.

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

From Table 4 above:

The amino acid sequence of Linker is shown in the preceding SEQ ID NO: 29:

Nucleic acid sequence of the heavy chain variable region H7v of H7L8 (360bp):

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

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

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

Amino acid sequence of the heavy chain of the immunoglobulin moiety in Bs-PL022B (hG1TM):

Nucleic acid sequence of the heavy chain of the immunoglobulin moiety in Bs-PL022B (hG1TM):

Amino acid sequence of the light chain of the immunoglobulin moiety in Bs-PL022B (hG1TM):

Nucleic acid sequence of the light chain of the immunoglobulin moiety in Bs-PL022B (hG1TM):

Based on 14C12H1L1, individual amino acids in the framework region (light chain) were mutated to obtain 14C12H1L1(M).

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

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

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

Experimental example 1: Fc segment mutation can effectively eliminate the anti-PD-1/CTLA4 bispecific anti-PD-1/CTLA4 immune checkpoint inhibitor. Body-mediated secretion of IL-8 and IL-6

1. Experimental materials:

Human peripheral monocyte derived macrophage (HPMM) is induced by human peripheral blood mononuclear cells (PBMC). The PBMCs used in this study were all isolated and prepared in Zhongshan Kangfang Biopharmaceutical Co., Ltd., and informed consent was obtained from the providers.

Ficoll-Paque ^™ PLUS Lymphocyte Separation Solution (GE, Cat. No.: 17-1440-03); RPMI 1640 (Gibco, Cat. No.: 22400-105); CHO-K1-PD1-CTLA4 cells (constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. ); FBS (Fetal Bovine Serum, Excell bio, Cat. No.: FSP500); Human IFN-γ protein (sinobio, Cat. No.: 11725-HNAS-100); LPS (Lipopolysaccharides), lipopolysaccharide (sigma, Cat. No.: L4391); 96-well Cell culture plates (Corning, Cat. No. 3599).

2. Experimental method:

Healthy human peripheral blood mononuclear cells (PBMC) were isolated according to the instructions of Ficoll-Paque ^TM Plus reagent for separation medium and resuspended in 1640 medium containing 2% FBS, and placed in a 37°C, 5% CO ₂ cell incubator. After 2 h, the supernatant was removed, the adherent cells were washed twice with PBS, and induced for 7 days by adding 1640 complete medium (containing 10% FBS) and 100 ng/mL human M-CSF. The medium was changed on days 3 and 5 and supplemented with M-CSF to induce HPMM. After HPMM induction was completed, the cells were collected, the number of HPMM cells was adjusted to 10,000/well, and IFN-γ (working concentration of 50 ng/mL) was added to the 96-well plate, and the total volume of each well was 100 μL. Collect CHO-K1 cells expressing human PD-1 and CTLA4, namely CHO-K1-PD1-CTLA4 cells, adjust the number of cells to 30,000/100μL/well; dilute the antibody with complete 1640 medium (working concentration: 25nM, 2.5 nM, 0.25nM), add 100 μL of antibody dilution to each well according to the experimental design, mix well, and design isotype control wells. Lipopolysaccharide (LPS) was used as a positive control drug, and the concentration was adjusted to 100 ng/mL from the complete medium in the experiment. The cell plates were placed in an incubator for 24 h. After centrifugation at 1200 rpm for 5 min, the supernatant was collected and the secretion of IL-8 and IL-6 was detected by Daktronics kit.

In this example, co-culture of CHO-K1-PD1-CTLA4 cells as target cells with HPMM can induce HPMM activation. After the activated HPMM is linked to the target cells through the antibody Fab, the Fc fragment of the antibody interacts with the FcγR on HPMM, causing HPMM to secrete cells factor.

3. Experimental results

As shown in Figure 1 and Figure 2.

The results showed that compared with wild-type IgG1 subtype antibody, the introduction of S228P mutation to improve stability of IgG4 subtype Nivolumab and IgG1 subtype Ipilimumab combined administration, and the anti-PD-1/CTLA4 bispecific with wild-type Fc segment Anti-PD-1/CTLA4 bispecific antibody (BiAb004(hG1TM)) carrying a mutation in the Fc segment can effectively eliminate the secretion of IL-6 and/or IL-8 by immune cells.

Experimental Example 2: Mutation of Fc segment can effectively eliminate PD-1/CD73 bispecific by bispecific immune checkpoint inhibitor Antibody-mediated secretion of IL-8 and IL-6

HPMM is induced by PBMC. The PBMCs used in this study were all isolated and prepared in Zhongshan Kangfang Biopharmaceutical Co., Ltd., and informed consent was obtained from the providers.

Ficoll-Paque ^TM PLUS Lymphocyte Separation Solution (GE, Item No.: 17-1440-03); RPMI 1640 (Gibco, Item No.: 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd.); U87-MG cells (cells from ATCC, purchased from Beijing Zhongyuan Leading Technology Co., Ltd.); FBS (Fetal Bovine Serum, Excell bio, product number: FSP500); human IFN-γ protein (sinobio, product number: 11725-HNAS-100) ; LPS (Lipopolysaccharides), lipopolysaccharide (sigma, Cat. No.: L4391); 96-well cell culture plate (Corning, Cat. No. 3599).

Healthy human PBMCs were isolated according to the instructions of the Ficoll-Paque ^™ Plus reagent for separation medium and resuspended in 1640 medium containing 2% FBS and placed in a 37°C, 5% CO ₂ cell incubator. After 2 h, the supernatant was removed, the adherent cells were washed twice with PBS, and induced for 7 days by adding 1640 complete medium (containing 10% FBS) and 100 ng/mL human M-CSF. The medium was changed on days 3 and 5 and supplemented with M-CSF to induce HPMM. On the 7th day, HPMM was collected after induction, and the concentration was adjusted to 100,000/mL with complete medium and dispensed into 96-well plates. Recombinant human IFN-γ (50ng/mL) was added, and the cells were incubated in an incubator for 24h. 24h later, the log-phase CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 were collected, and the concentration was adjusted to 300,000/mL with complete medium after resuspending. Antibodies were diluted in complete medium to working concentrations of 25nM, 2.5nM, 0.25nM. At the same time, an isotype control antibody and a blank control were designed. Remove the supernatant from the 96-well plate, add CHO-K1-PD1 or U87-MG cell suspension and antibody (final volume 200 μL), mix well, and incubate in an incubator for 24 h. After centrifugation at 500×g for 5 min, the supernatant was collected and the secretion of IL-8 and IL-6 was detected by Daktronics kit. LPS was used as a positive control drug, and the concentration was adjusted to 100 ng/mL from the complete medium in the experiment.

In this example, co-culture of CHO-K1-PD1 and U87-MG cells as target cells with HPMM can induce HPMM activation. After the activated HPMM is linked to the target cells through the antibody Fab, the antibody Fc segment interacts with the FcγR on HPMM, causing HPMM secretes cytokines.

3. Experimental results

As shown in Figure 3 to Figure 6.

The results show that, compared with the wild-type IgG1 subtype PD-1 antibody or CD73 antibody, the anti-PD-1/CD73 bispecific antibody carrying the Fc segment mutation can effectively eliminate the IL-6 and/or IL of immune cells. -8 secretion.

In addition, compared with the wild-type bispecific antibody NTPDV2 (hG1WT) without Fc mutation, the anti-PD-1/CD73 bispecific antibody with Fc mutation can also effectively eliminate IL-6 and IL-6 from immune cells. /or secretion of IL-8.

Experimental Example 3: Mutation of the Fc segment can effectively eliminate the anti-PD-1/LAG3 bispecific anti-PD-1/LAG3 immune checkpoint inhibitor. Body-mediated secretion of IL-8 and IL-6

1. Experimental materials:

Ficoll-Paque ^TM PLUS Lymphocyte Separation Solution (GE, Cat. No.: 17-1440-02); RPMI 1640 (Gibco, Cat. No.: 22400-105); CHO-K1-PD1-LAG3 cells (constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. ); FBS (Fetal Bovine Serum, Excell bio, Cat. No.: FSP500); Human IFN-γ protein (sinobio, Cat. No.: 11725-HNAS-100); LPS (Lipopolysaccharides, sigma, Cat. No.: L6529); 96-well cell culture plate ( Corning, Cat. No. 3599).

2. Experimental method:

Healthy human peripheral blood mononuclear cells (PBMC) were isolated according to the instructions of Ficoll-Paque ^TM Plus reagent for separation medium and resuspended in 1640 medium containing 2% FBS, and placed in a 37°C, 5% CO ₂ cell incubator. After 2 h, the supernatant was removed, the adherent cells were washed twice with PBS, and induced for 7 days by adding 1640 complete medium (containing 10% FBS) and 100 ng/mL human M-CSF. The medium was changed on days 3 and 5 and supplemented with M-CSF to induce HPMM. After HPMM induction was completed, the cells were collected, the number of HPMM cells was adjusted to 10,000/well, and IFN-γ (working concentration of 50 ng/mL) was added to a 96-well plate for a total of 120 wells, and the total volume of each well was 100 μL. Collect CHO-K1 cells expressing human PD-1 and LAG3, namely CHO-K1-PD1-LAG3 cells, adjust the number of cells to 30,000/100μL/well; dilute the antibody with 1640 complete medium (working concentration: 25nM, 2.5 nM, 0.25nM), add 100 μL of antibody dilution to each well according to the experimental design, mix well, and design isotype control wells. Lipopolysaccharide (LPS) was used as a positive control drug, and the concentration was adjusted to 100 ng/mL from the complete medium in the experiment. The cell plates were placed in an incubator for 24 h. The cell plate was taken out and centrifuged at 1200 rpm for 5 min, the supernatant was collected, and the secretion of IL-8 and IL-6 was detected by Daktronics kit.

In this example, co-culture of CHO-K1-PD1-LAG3 cells as target cells with HPMM can induce HPMM activation. After the activated HPMM is linked to the target cells through the antibody Fab, the antibody Fc fragment interacts with the FcγR on HPMM, causing HPMM to secrete cells factor.

3. Experimental results

As shown in Figure 7 and Figure 8.

The results showed that compared with the wild-type IgG1 subtype antibody, Nivolumab of the IgG4 subtype that introduced the S228P mutation to improve the stability, Relatlimab of the IgG1 subtype, the anti-PD-1 antibody 14C12H1L1 (hG1WT) without the Fc segment mutation, and the wild The anti-LAG3 antibody H7L8 (hG1WT) with Fc-type Fc-segment and the anti-PD-1/LAG3 bispecific antibody (Bs-PL022B(hG1TM)) carrying a mutation in the Fc-segment can effectively eliminate IL-6 and/or IL from immune cells -8 secretion.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions of those details may be made within the scope of the present invention in light of all the teachings disclosed. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

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

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

L234A and L235A;

L234A and G237A;

L235A and G237A;

or

L234A, L235A and G237A.
The method of claim 1, wherein the drug containing an immunoglobulin Fc fragment comprises an antibody and/or an Fc fusion protein;

Optionally, the medicine containing the immunoglobulin Fc fragment further comprises one or more pharmaceutically acceptable excipients.
The method of claim 2, wherein the antibody is an immune checkpoint inhibitor.
The method of any one of claims 1 to 3, wherein the antibody is a bispecific antibody or a multispecific antibody.
The method of any one of claims 1-4, wherein the antibody targets:

PD-1 and CTLA4, PD-1 and CD73, PD-1 and LAG3, CTLA4 and CD73, CTLA4 and LAG3, or CD73 and LAG3.
The method of any one of claims 4-5, wherein the bispecific antibody targets PD-1 and CTLA4, comprising:

targeting the first protein domain of PD-1, and

Targets the second protein functional region of CTLA4;

in:

The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; wherein, the immunoglobulin, its heavy chain variable region comprises amino acid sequences such as SEQ ID NOs: 32- HCDR1-HCDR3 shown in 34, its light chain variable region comprises the LCDR1-LCDR3 shown in amino acid sequence respectively as SEQ ID NOs:35-37; And described single chain antibody, its heavy chain variable region comprises amino acid sequence HCDR1-HCDR3 as shown in SEQ ID NOs:38-40 respectively, the light chain variable region of which comprises LCDR1-LCDR3 as shown in SEQ ID NOs:41-43 respectively with amino acid sequence;

or,

The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; wherein, the immunoglobulin, its heavy chain variable region comprises amino acid sequences such as SEQ ID NOs:38- HCDR1-HCDR3 shown in 40, the light chain variable region of which comprises the LCDR1-LCDR3 amino acid sequences shown in SEQ ID NOs: 41-43 respectively; and the single-chain antibody, whose heavy chain variable region comprises the amino acid sequence HCDR1-HCDR3 as shown in SEQ ID NOs:32-34 respectively, the light chain variable region of which comprises the LCDR1-LCDR3 as shown in SEQ ID NOs:35-37 respectively with amino acid sequence;

the immunoglobulin is human IgG; and

There are two single-chain antibodies, and one end of each single-chain antibody is respectively connected to the C-terminus of the two heavy chains of immunoglobulin.
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 light chain variable region of the immunoglobulin is selected from 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 SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31;

or,

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: 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 the group consisting of SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO:64.
The method according to claim 6 or 7, wherein the bispecific antibody is selected from any one of the following (1)-(18):

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

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

(7)

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

(8)

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

(9)

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

(10)

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

(11)

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

(12)

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

(13)

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

(14)

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

(15)

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

(16)

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

(17)

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

(18)

The amino acid sequence of the variable region of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 30, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is shown in SEQ ID NO: 31; and, the The amino acid sequence of the variable region of the heavy chain of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is shown in SEQ ID NO: 64.
The method of any one of claims 4-5, wherein the bispecific antibody targets CD73 and PD-1 comprising:

targeting the first protein domain of CD73, and

Targeting the second protein functional region of PD-1;

in:

The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; wherein, the immunoglobulin, its heavy chain variable region comprises amino acid sequences such as SEQ ID NOs:44- HCDR1-HCDR3 shown in 46, its light chain variable region comprises the LCDR1-LCDR3 shown in amino acid sequence as SEQ ID NOs:47-49 respectively; And described single chain antibody, its heavy chain variable region comprises amino acid sequence HCDR1-HCDR3 as shown in SEQ ID NOs:32-34 respectively, the light chain variable region of which comprises the LCDR1-LCDR3 as shown in SEQ ID NOs:35-37 respectively with amino acid sequence;

or,

The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; wherein, the immunoglobulin, its heavy chain variable region comprises amino acid sequences such as SEQ ID NOs: 32- HCDR1-HCDR3 shown in 34, its light chain variable region comprises the LCDR1-LCDR3 shown in amino acid sequence respectively as SEQ ID NOs:35-37; And described single chain antibody, its heavy chain variable region comprises amino acid sequence HCDR1-HCDR3 as shown in SEQ ID NOs:44-46 respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:47-49 respectively with amino acid sequence;

the immunoglobulin is human IgG; and

There are two single-chain antibodies, and one end of each single-chain antibody is respectively connected to the C-terminus of the two heavy chains of immunoglobulin.
The method of claim 9, wherein,

The amino acid sequence of the variable region of the heavy chain 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 SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region of the 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: 6; NO:8 and SEQ ID NO:64;

or,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from 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 light chain variable region of the single chain antibody The amino acid sequence is selected from SEQ ID NO:26.
The method according to claim 9 or 10, wherein the bispecific antibody is selected from any one of the following (1)-(6):

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

The amino acid sequence of the variable region of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is shown in SEQ ID NO: 64; and, the The amino acid sequence of the variable region of the heavy chain of the single chain antibody is shown in SEQ ID NO: 22, and the amino acid sequence of the variable region of the light chain of the single chain antibody is shown in SEQ ID NO: 26.
The method of claim 4 or 5, wherein the bispecific antibody targets LAG3 and PD-1 comprising:

targeting the first protein domain of LAG3, and

Targeting the second protein functional region of PD-1;

in:

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

or,

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

the immunoglobulin is human IgG; and

There are two single-chain antibodies, and one end of each single-chain antibody is respectively connected to the C-terminus of the two heavy chains of immunoglobulin.
The method of claim 12, wherein,

The amino acid sequence of the variable region of the heavy chain 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 SEQ ID NO: 59; The amino acid sequence of the heavy chain variable region of the 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: 6; NO:8 and SEQ ID NO:64;

or,

The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 4 , SEQ ID NO: 8 and SEQ ID NO: 64; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 57; and the light chain variable region of the single chain antibody The amino acid sequence is selected from SEQ ID NO:59.
The method according to claim 12 or 13, wherein the bispecific antibody is selected from any one of the following (1)-(6):

(1)

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

(2)

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

(3)

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

(4)

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

(5)

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

(6)

The amino acid sequence of the variable region of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is shown in SEQ ID NO: 64; and, the The amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:57, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:59.
The method of any one of claims 6 to 14, wherein the heavy chain constant region of the immunoglobulin is selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4, and the immunoglobulin The light chain constant region is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4;

Preferably, the heavy chain constant region of the immunoglobulin is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region of the immunoglobulin is a human Ig kappa chain C region.
The method of any one of claims 1 to 15, wherein the immune cells are human immune cells, such as human macrophages.
17. The method of any one of claims 1 to 16, wherein the method is a method for non-therapeutic purposes.
The method of any one of claims 1 to 17, wherein the method is a method for pharmaceutical purposes.
A method for improving the efficacy and/or safety of a drug containing an immunoglobulin Fc fragment, wherein the drug containing an immunoglobulin Fc fragment is reduced by the method of any one of claims 1 to 18 Mediated levels of IL-8 and/or IL-6 secreted by immune cells.