Anti-CTLA4 / anti-PD-1 bispecific antibodies and uses thereof

Abstract

The present invention relates to the fields of tumor therapy and molecular immunology, and in particular to an anti-CTLA4 / anti-PD-1 bispecific antibody and its use. In particular, the anti-CTLA4 / anti-PD-1 bispecific antibody comprises a first protein functional region targeting PD-1 and a second protein functional region targeting CTLA4, wherein the heavy chain constant region of the immunoglobulin contained in the bispecific antibody has mutations at any two or three of positions 234, 235, and 237 according to the EU numbering system, and the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced after the mutations compared to that before the mutations. The bispecific antibody of the present invention can bind well and specifically to CTLA4 and PD-1, specifically relieve the immunosuppression of CTLA4 and PD-1 in an organism, and activate T lymphocytes, thereby showing promising applications.

Description

[Technical Field]

[0001] The present invention relates to the fields of tumor therapy and molecular immunology, in particular to anti-CTLA4 / anti-PD-1 bispecific antibodies and uses thereof. More particularly, the present invention relates to mutant anti-CTLA4 / anti-PD-1 bispecific antibodies. [Background technology]

[0002] The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. Both PD-1 ligands, PDL1 (programmed cell death 1 ligand 1, or PDL-1) and PDL2 (programmed cell death 1 ligand 2, or PDL-2), are members of the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only on antigen-presenting cells, such as dendritic cells and macrophages.

[0003] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microorganism infection, and tumor immune evasion. PD-1 is primarily expressed in immune cells, such as T cells, and its ligand, PDL1, is highly expressed in several human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway activates inhibited T cells, allowing them to attack cancer cells. Blocking PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, activate tumor cell killing processes, and further inhibit local tumor growth (Julie R et al., 2012, N Engl J Med., 366:2455-2465). Additionally, tumors with high PDL1 expression are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA, 104:3360-5). An effective method is to administer anti-PD-1 antibodies to modulate the expression of PD-1. Due to the broad anti-tumor promise and surprising efficacy of PD-1 antibodies, it is widely recognized in the industry that antibodies targeting the PD-1 pathway will bring about a breakthrough in the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol., 42(3):466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets., 13(7):768-74).

[0004] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules are highly similar in terms of gene structure, chromosomal location, sequence homology, and gene expression. Both molecules are receptors for the costimulatory molecule B7, which is expressed primarily on the surface of activated T cells. Binding of CTLA4 to B7 inhibits activation of mouse and human T cells and plays a negative regulatory role in T cell activation.

[0005] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing T cell responsiveness to various antigens. In this regard, in vivo and in vitro studies are essentially consistent. Currently, there are CTLA4 monoclonal antibodies in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun., 13:5).

[0006] Interleukin-2 (IL-2) is produced by T cells. It is a growth factor that regulates T cell subpopulations and is an important factor in regulating immune responses. It promotes the proliferation of activated B cells and participates in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the U.S. FDA for the treatment of malignant tumors, including melanoma and renal cancer, and clinical studies are currently underway for the treatment of chronic viral infections (Chavez, AR, et al., 2009, Ann. NY Acad. Sci., 1182:14-27). CTLA4 and CTLA4 antibodies are important influencers of T cell function and interfere with the immune microenvironment throughout the body. In vitro and in vivo studies have demonstrated that CTLA4 antibodies specifically alleviate CTLA4 immunosuppression, activate T cells, and induce IL-2 production, suggesting promising applications in gene therapy for diseases such as tumors and parasitic infections.

[0007] CTLA4 antibodies can produce unique therapeutic effects and remarkable efficacy against diseases and can be used to complement traditional medicines and to explore new avenues of gene therapy.

[0008] Bispecific antibodies, also known as bifunctional antibodies, are unique drugs that simultaneously target two different antigens and can be produced by immunomagnetic separation. Alternatively, they can be obtained by genetic engineering. Genetic engineering has certain advantages, as it allows flexibility in terms of binding site optimization, synthesis form, yield, and the like. Currently, more than 45 forms have been demonstrated (Dafne Muller, Kontermann R E., 2010, BioDrugs, 24(2):89-98). Many developed bispecific antibodies are in the form of IgG-scFv, i.e., the Morrison format (Coloma MJ, Morrison SL., 1997, Nat Biotechnol., 15:159-163), which has been demonstrated to be one of the ideal formats for bispecific antibodies due to its similarity to the naturally occurring IgG format and advantages in antibody engineering, expression, and purification (Miller BR, Demarest SJ, et al., 2010, Protein Eng Des Sel, 23:549-57; Fitzgerald J, Lugovskoy A., 2011, MAbs, 3:299-309).

[0009] ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the killing of target cells by killer cells (NK cells, macrophages, etc.) mediated by the binding of the Fab fragment of the antibody to an epitope on a virus-infected cell or tumor cell and the binding of the Fc fragment of the antibody to an Fc receptor (FcR) on the surface of the killer cell.

[0010] CDC (complement-dependent cytotoxicity) refers to the specific binding of an antibody to a corresponding antigen on the surface of a cell membrane, forming a complex that activates the complement system, which further forms MAC on the surface of the target cell, resulting in subsequent lysis of the target cell. Complement can cause the lysis of various bacteria and other pathogenic organisms and is an important defense mechanism against infection by pathogenic organisms.

[0011] Fc receptors belong to the immunoglobulin family and are expressed on the surface of specific immune cells to recognize the Fc region of antibodies and mediate immune responses. After the Fab region recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (e.g., killer cell) to initiate immune cell response functions, such as phagocytosis and ADCC.

[0012] Fc receptors are primarily classified into three types: FcγR, FcαR, and FcεR, according to the type of antibody recognized by the Fc receptor and the type of cell on which they are expressed. FcγR can be further classified into four subtypes: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcRn (neonatal Fc receptor). Among these, FcγRI, FcγRII, and FcγRIII are closely associated with ADCC effects. FcγRIII is the most predominant molecule mediating ADCC and has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. Within the FcγRIIIa population, there are two subtypes, FcγRIIIa_V158 with high affinity and FcγRIIIa_F158 with low affinity, distinguished by a single nucleotide polymorphism (SNP) site. FcγRI has higher affinity for the Fc region of IgG and participates in the ADCC process; FcγRII includes three subtypes, FcγRIIa, FcγRIIb, and FcγRIIc (also called CD32a, CD32b, and CD32c, respectively), among which FcγRIIa has ADCC activity; FcγRIIa has two subtypes, FcγRIIa_H131 and FcγRIIa_R131, due to a single nucleotide mutation in humans; FcγRIIb is an inhibitory receptor and a typical inhibitory FcγR that inhibits the nearby ITAM pathway. For example, after binding of an immune complex to the BCR, the Fc fragment binds to FcγRIIb on the same cell, negatively regulating B cell activation and reducing antibody and cytokine secretion (Hogarth PM, Pietersz GA., 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).

[0013] The IgG family includes four members, IgG1, IgG2, IgG3, and IgG4, which differ in amino acids in the fragment crystallizable (Fc) region of the heavy chain constant region, resulting in their different affinities for FcγR. IgG1 is the most abundant subtype in humans and is also the most common subtype used in monoclonal antibody pharmaceuticals. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the lowest affinity for FcγRs but can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 is characterized by the highest binding ability to FcγRs and can induce ADCC and a higher CDC effect than IgG1. IgG4 molecules demonstrate weaker binding to FcγRs other than FcγRI and have a lower probability of inducing CDC and NK cell-mediated ADCC. However, antibodies of the IgG4 subtype can mediate the ADCP effect through binding to FcγRI, and the ADCP effect present in antibody therapies targeting immune cells can cause damage to the immune cells, leading to adverse pharmacological effects. Currently, there remains a need to develop novel anti-CTLA4 / anti-PD-1 bispecific antibodies to reduce or eliminate the damage caused by antibody-mediated ADCC, ADCP, and / or CDC activity against immune cells to which the anti-CTLA4 / anti-PD-1 bispecific antibodies bind, and to improve the efficacy of antibody therapies.

[0014] Chemotherapy is currently classified into nine main classes (He Jie et al., Clinical Oncology, Beijing, People's Medical Publishing House, 2016:230-237). The first class is a class of drugs that directly bind to DNA and prevent DNA replication, including various alkylating agents, mitomycin, bleomycin, dacarbazine, platinum-based drugs (e.g., cisplatin and carboplatin), camptothecin, and their derivatives. The second class is a class of drugs that prevent nucleic acid biosynthesis, primarily affecting the enzyme systems of tumor cells to block the synthesis of DNA and RNA precursors, thereby inhibiting the formation of DNA or RNA, including methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea, and cytarabine; such drugs primarily act on cells in the S phase and are antimetabolite chemotherapy agents and cell cycle-specific anticancer drugs. The third class is chemotherapy drugs that affect transcription through a pharmacological mechanism in which the drug intercalates into the DNA double helix, forming non-covalent bonds with the DNA double helix and interfering with the transcription of genetic information on DNA into DNA-dependent mRNA, resulting in impaired template function and disrupted transcription. The fourth class is drugs that affect tubulin and mitosis, including vinca alkaloids, podophyllotoxins, and taxanes. The fifth class is drugs that affect ribosomal function to block protein synthesis; a representative example of such a drug is harringtonine, which inhibits the initiation of protein synthesis, disassembling ribosomes and releasing new peptide chains, but does not block the binding of mRNA and tRNA to ribosomes; such drugs cause the reduction of nuclear DNA and cytoplasmic RNA and the depolymerization of polysomes, inhibiting mitosis. The sixth class is drugs that affect the membranes of tumor cells, such as concanavalin A (Con-A) and phytohemagglutinin (PHA), which bind to glycoprotein receptors on the cell membrane, thereby affecting DNA synthesis in tumor cells and preventing them from dividing. The seventh class is drugs that induce apoptosis, such as arsenic trioxide.The eighth class are hormones that treat tumors by modulating the endocrine system, including estrogens, antiestrogens, progestogens, androgens, antiandrogens, corticosteroids, and anticorticosteroids (including dichlorodiphenyldichloroethane and aminoglutethimide). The ninth class are targeted anti-cancer therapies, including monoclonal antibodies, epidermal growth factor signaling inhibitors (e.g., drugs targeted to receptor tyrosine kinase pathways), ubiquitin-proteasome inhibitors, and angiogenesis inhibitors.

[0015] Anlotinib is a quinoline derivative tyrosine kinase inhibitor. As a multitarget tyrosine kinase inhibitor (TKI), it affects tumor angiogenesis and proliferation signaling. Primary targets include the receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFR) 1-3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptors (FGFR) 1-4, platelet-derived growth factor receptors (PDGFR) α and β, and stem cell factor receptors (SCFR) 7, 8, and 9. A phase 2 trial showed that anlotinib improved progression-free survival (PFS) with a potential benefit for overall survival (Han B, et al., Br J Cancer, 2018; 118(5):654-661). A multicenter, double-blind, randomized phase 3 clinical trial demonstrated that anlotinib resulted in prolonged and progression-free survival in Chinese patients. The findings suggested that anlotinib was well tolerated and a potential third-line or additional treatment for patients with advanced NSCLC (Han B, et al., JAMA Oncol., 2018 Nov.; 4(11):1569-1575).

[0016] Example 24 of patent document WO 2008112407 discloses a quinoline-derived tyrosine kinase inhibitor 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropylamine and a method for preparing the same. The structural formula of the quinoline-derived tyrosine kinase inhibitor is shown in Formula I. Anlotinib hydrochloride is the hydrochloride salt of the compound of Formula I. [ka] Lenvatinib, an oral multiple tyrosine kinase inhibitor developed by Eisai (Japan), is a multitargeted receptor tyrosine kinase inhibitor that inhibits the kinase activity of VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4). In addition to normal cellular functions, lenvatinib also inhibits other receptor tyrosine kinases involved in pathogenic angiogenesis, tumor growth, and cancer progression, including the fibroblast growth factor (FGF) receptors FGFR1, FGFR2, FGFR3, and FGFR4, the "rearranged during transfection" (RET) receptor, KIT, and platelet-derived growth factor receptor α (PDGFRα). Lenvatinib also exhibits antiproliferative activity in hepatocellular carcinoma cell lines, which is dependent on the activation of FGFR signaling and the simultaneous inhibition of FGF receptor substrate 2α (FRS2α) phosphorylation.

[0017] The structure of lenvatinib, 4-(3-chloro-4(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, is disclosed in Example 368 of U.S. Patent No. 7,612,208. U.S. Patent No. 7,253,286 discloses a mesylate form of lenvatinib (i.e., lenvatinib mesylate), named 4-[3-chloro-4-(cyclopropylureido)phenoxy]-7-methoxyquinoline-6-carboxamide mesylate, the chemical structure of which is provided below (Formula II): [ka] However, for various tumors, the disease is still not controllable for a long time after chemotherapy, and the 5-year survival rate is still very low. Therefore, the development of drugs or combination therapies with lower toxicity and higher efficacy is of great significance. Summary of the Invention

[0018] Through intensive research and creative efforts, the inventors have correspondingly modified the Fc fragment of the anti-CTLA4 / anti-PD-1 antibody structure to reduce the binding ability of the Fc region to Fc receptors, thereby reducing the ADCC, ADCP and / or CDC effects on immune cells and increasing the efficacy of the anti-CTLA4 / anti-PD-1 antibody.

[0019] The invention is described in detail below.

[0020] One embodiment of the present invention is a bispecific antibody comprising: a first protein functional region that targets PD-1; and a second protein functional region that targets CTLA4; 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; For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; Or, For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the immunoglobulin is a human IgG1 subtype immunoglobulin; the heavy chain constant region of the immunoglobulin has mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced after the mutations compared to that before the mutations; preferably, the affinity constant is measured by a Fortebio Octet system. This relates to bispecific antibodies.

[0021] In one or more embodiments of the invention, for bispecific antibodies, after the above-mentioned mutations, the affinity constant of the bispecific antibody for FcγRIIIa, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb is reduced compared to that before the mutations; preferably, the affinity constant is measured using a Fortebio Octet system.

[0022] In one or more embodiments of the invention, for bispecific antibodies, the heavy chain constant region of the immunoglobulin comprises the following mutations at positions 234, 235 and / or 237 according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A It has.

[0023] In the present invention, unless otherwise specified, the letter before the position number represents the amino acid before mutation, and the letter after the position number represents the amino acid after mutation.

[0024] The present invention further provides a bispecific antibody comprising: a first protein functional region that targets PD-1; and a second protein functional region that targets CTLA4; 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; For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; Or, For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the immunoglobulin is a human IgG1 subtype immunoglobulin; The heavy chain constant region of the immunoglobulin has the following mutations at positions 234, 235 and / or 237 according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A having This relates to bispecific antibodies.

[0025] In one or more embodiments of the invention, for bispecific antibodies, the heavy chain constant region of the immunoglobulin according to the EU numbering system is N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A The gene has one or more mutations selected from:

[0026] In one or more embodiments of the invention, the bispecific antibody is in the form of an IgG-scFv, i.e., Morrison format.

[0027] In one or more embodiments of the invention, for a bispecific antibody: The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:14 and SEQ ID NO:18; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:16 and SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 and SEQ ID NO:43; the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:44; Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 and SEQ ID NO:43; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:44; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:14 and SEQ ID NO:18; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:16 and SEQ ID NO:20.

[0028] In one or more embodiments of the present invention, the bispecific antibody comprises any one of the following (1) to (20): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 4; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 8; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 12; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 4; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 8; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 12; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:16; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:20; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:16; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:20; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 42; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 42; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; and, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20. is selected from one of the following:

[0029] In one or more embodiments of the invention, for a bispecific antibody: The amino acid sequence of the immunoglobulin heavy chain is set forth in SEQ ID NO:40, and the amino acid sequence of the immunoglobulin light chain is set forth in SEQ ID NO:24.

[0030] In one or more embodiments of the invention, for bispecific antibodies, the immunoglobulin or antigen-binding fragment thereof is about 10 -7 Higher than M, e.g., about 10 -6 M, 10 -5 M, 10 -4 M or 10-3 binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb with an affinity constant greater than or equal to M; preferably, the affinity constant is measured using a Fortebio Octet system; Preferably, the immunoglobulin or antigen-binding fragment thereof has no binding signal or a binding signal lower than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the binding signal refers to the response measured by a Fortebio Octet system.

[0031] In one or more embodiments of the invention, for bispecific antibodies, the immunoglobulin or antigen-binding fragment thereof is about 10 -9 Higher than M, e.g., about 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 binds to C1q with an affinity constant greater than or equal to M; preferably, the affinity constant is measured by a Fortebio Octet system; Preferably, the immunoglobulin or antigen-binding fragment thereof has no binding signal to C1q or a binding signal below 0.1 nm; preferably, the binding signal refers to the response measured by the Fortebio Octet system.

[0032] In one or more embodiments of the invention, for bispecific antibodies, a first protein functional region is linked, either directly or via a linker fragment, to a second protein functional region; and / or a heavy chain variable region of a single chain antibody is linked, either directly or via a linker fragment, to a light chain variable region of a single chain antibody.

[0033] In one or more embodiments of the invention, for bispecific antibodies, the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5, or 6.

[0034] In one or more embodiments of the invention, for a bispecific antibody, the number of first protein functional regions and second protein functional regions is independently one, two or more.

[0035] In one or more embodiments of the invention, for a bispecific antibody, the number of first protein functional regions is one and the number of second protein functional regions is two.

[0036] In one or more embodiments of the present invention, for bispecific antibodies, the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since an immunoglobulin has two heavy chains, two single-chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain antibody molecules are identical. Preferably, the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin by forming an amide bond via the linker fragment described above.

[0037] In one or more embodiments of the invention, the constant regions of the immunoglobulins are humanized, for example, the heavy chain constant region is the Ig gamma-1 chain C region, Accession: P01857, and the light chain constant region is the Ig kappa chain C region, Accession: P01834.

[0038] In one or more embodiments of the invention, the bispecific antibody has a -5 Lower than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Lower than M or lower K D It binds to the CTLA4 protein and / or the PD-1 protein.

[0039] In one or more embodiments of the invention, the bispecific antibody is a monoclonal antibody.

[0040] In one or more embodiments of the invention, the bispecific antibody is a humanized antibody.

[0041] In one or more embodiments of the invention, the bispecific antibody comprises: a first protein functional region that targets PD-1; and A second protein functional domain that targets CTLA4 Includes; the number of first protein functional regions is one and the number of second protein functional regions is two; the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; The amino acid sequence of the immunoglobulin heavy chain is set forth in SEQ ID NO: 40, and the amino acid sequence of the immunoglobulin light chain is set forth in SEQ ID NO: 24; The amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; Single chain antibodies are linked to the C-terminus of the immunoglobulin heavy chain; the first protein functional region is linked to the second protein functional region via a first linker fragment; and the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 25 and SEQ ID NO: 26; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:26.

[0042] Another aspect of the invention pertains to an isolated nucleic acid molecule encoding a bispecific antibody according to any embodiment of the invention.

[0043] The present invention also relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0044] The present invention also relates to a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention.

[0045] Another aspect of the invention relates to a conjugate comprising an antibody or antigen-binding fragment thereof and a conjugated moiety, wherein the immunoglobulin is a bispecific antibody according to any embodiment of the invention, and the conjugated moiety is a detectable label; preferably, the conjugated moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0046] Another aspect of the invention is a kit comprising a bispecific antibody according to any embodiment of the invention or comprising a conjugate of the invention; Preferably, the kit further comprises a second antibody capable of specifically recognizing the immunoglobulin or an antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, for example, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. Regarding the kit.

[0047] Another aspect of the invention relates to the use of a bispecific antibody or conjugate according to any embodiment of the invention in the preparation of a kit for detecting the presence or level of PD-1 and / or CTLA4 in a sample.

[0048] Another aspect of the invention relates to a pharmaceutical composition comprising a bispecific antibody or conjugate according to any embodiment of the invention; optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

[0049] In one or more embodiments of the present invention, the pharmaceutical composition further comprises one or more anti-tumor chemotherapeutic agents; Preferably, the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor; more preferably, the antitumor chemotherapeutic agent is anlotinib or a pharmaceutically acceptable salt thereof (e.g., the hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., the mesylate salt).

[0050] In one or more embodiments of the present invention, the unit dose of the pharmaceutical composition is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg based on the mass of the bispecific antibody.

[0051] Another aspect of the present invention is a combination product comprising a first product and a second product in separate packages, a first article of manufacture comprising a bispecific antibody, conjugate or pharmaceutical composition according to any embodiment of the invention; The second product comprises one or more anti-tumor chemotherapeutic agents; preferably, the anti-tumor chemotherapeutic agent is a tyrosine kinase inhibitor; more preferably, the anti-tumor chemotherapeutic agent is anlotinib or a pharmaceutically acceptable salt thereof (e.g., the hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., the mesylate salt); Preferably, the first product and the second product independently further comprise one or more pharmaceutically acceptable excipients; Preferably, the combination product further comprises a package insert. Regarding combination products.

[0052] In one or more embodiments of the invention, for combination products, the unit dose of the first product is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg based on the mass of the bispecific antibody.

[0053] In one or more embodiments of the invention, for combination products, the unit dose of the second product is 0.1 to 100 mg, 0.5 to 50 mg, 0.5 to 10 mg, 1 to 10 mg, 2 to 8 mg, or 1 to 5 mg based on the mass of the active ingredient.

[0054] In one or more embodiments of the invention, for combination products, the unit dose of the second product is 1 to 20 mg, 2 to 15 mg, 4 to 12 mg, or 8 to 12 mg based on the mass of the active ingredient.

[0055] Another aspect of the invention is the use of a bispecific antibody, a conjugate, a pharmaceutical composition or a combination product according to any embodiment of the invention in the preparation of a medicament for treating and / or preventing a tumor or anemia, or for diagnosing a tumor or anemia; preferably wherein the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma or ovarian germ cell neoplasm of MSI-H / dMMR phenotype; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. Regarding use.

[0056] MSI refers to microsatellite instability. Microsatellites are short tandem repeats throughout the human genome, containing 10 to 50 repeats of one, two, or more nucleotides. Microsatellites in certain abnormal cells, such as tumors, are altered in length by the insertion or deletion of repeat units compared to normal cells. Such alterations are called MSI. Based on the level and extent of instability, MSI can be classified as microsatellite instability-high (MSI-H), microsatellite instability-low (MSI-L), and microsatellite stability (MSS). The primary cause of MSI is DNA mismatch repair (MMR). Human mismatch repair genes (MMR genes) can express corresponding mismatch repair proteins through transcription and translation. The absence of any MMR protein can lead to mismatch repair deficiency, which causes base pair mismatches to accumulate during the DNA replication process, ultimately resulting in MSI. Approximately 15% of colorectal cancers are caused by the MSI pathway. This was first reported in colorectal cancer, but can also occur in gastric cancer, endometrial cancer, adrenocortical carcinoma, and the like (Baretti M et al., Pharmacol Ther., 2018; 189:45-62). Subsequent studies have also found MSI-H / dMMR signatures in mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell neoplasms.

[0057] MSI-H and dMMR represent the results of two different assays, which are biologically consistent and are referred to as MSI-H / dMMR or frequent MSI / dMMR, while MSI-L and MSS are phenotypes of proficiency MMR (pMMR). dMMR detection involves performing immunohistochemistry for protein expression of four mismatched genes, MSH2, MLH1, MSH6, and PMS2, on tumor samples (including surgical and aspirate specimens). Absence of any of the four proteins confirms dMMR; positive results for all four proteins indicate pMMR, i.e., intact mismatch repair function. MSI detection involves matching and comparing the lengths of repeated DNA sequences (microsatellite sequences) in tumor cells and somatic cells. When five standard loci are detected using PCR based on the American NCI standard, inconsistency at two or more loci indicates instability, defined as MSI-H, one inconsistent locus indicates MSI-L, and five consistent loci indicate MSS. High-throughput sequencing (also called next-generation sequencing, or NGS) can also be used as a method to detect microsatellite instability. When more microsatellite loci are selected, such as more than five loci or other microsatellite loci, for the PCR assay, inconsistency at ≥ 30% of loci is defined as MSI-H, consistency at all loci is defined as MSS, and inconsistency between 0 and 30% is defined as MSI-L.

[0058] Another aspect of the present invention is a method for producing a semiconductor device comprising: a pharmaceutical agent for blocking the binding of PD-1 to PD-L1; a pharmaceutical agent for down-regulating the activity or level of PD-1; a drug for alleviating the immunosuppression of PD-1 in an organism; or A pharmaceutical agent for increasing the expression of IFN-γ and / or IL-2 in T lymphocytes; and / or a pharmaceutical agent for blocking the binding of CTLA4 to B7; a pharmaceutical agent for down-regulating the activity or level of CTLA4; a medicament for alleviating immunosuppression of CTLA4 in an organism, or Drug for increasing IL-2 expression in T lymphocytes The present invention relates to the use of a bispecific antibody or conjugate according to any embodiment of the invention in the preparation of a

[0059] Another aspect of the invention is a method for the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of a tumor, comprising administering to a subject in need thereof an effective amount of a bispecific antibody, conjugate, pharmaceutical composition or combination product according to any embodiment of the invention; preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasms selected from one or more of: Regarding the method.

[0060] In one or more embodiments of the invention, for the methods, administration occurs before or after surgery and / or before or after radiation therapy.

[0061] In one or more embodiments of the invention, the method comprises: The unit dose of the anti-CTLA4 / anti-PD-1 bispecific antibody is 0.1 to 100 mg per kg of body weight, preferably 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg); alternatively, the unit dose of the anti-CTLA4 / anti-PD-1 bispecific antibody is 10 to 1000 mg (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg), preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg, for each subject; Preferably, the dose is given once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks; Preferably, the route of administration is intravenous infusion or injection.

[0062] In some embodiments, administration of the anti-CTLA4 / anti-PD-1 bispecific antibody is in a 2-week (14 day) or 3-week (21 day) cycle, and preferably the anti-CTLA4 / anti-PD-1 bispecific antibody is administered intravenously on day 1 (D1) of each cycle. For example, the anti-CTLA4 / anti-PD-1 bispecific antibody is administered once every 2 weeks (q2w) or 3 weeks (q3w).

[0063] 1. A bispecific antibody, conjugate, pharmaceutical composition or combination product according to any embodiment of the invention for use in the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of a tumor; preferably wherein the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasms selected from one or more of: There is also provided a bispecific antibody, a conjugate, a pharmaceutical composition or a combination product according to any embodiment of the invention for said use.

[0064] blocking the binding of PD-1 to PD-L1; downregulating PD-1 activity or levels; alleviating PD-1 immunosuppression in the organism, or increasing IFN-γ expression in T lymphocytes; and / or blocking the binding of CTLA4 to B7; downregulating the activity or levels of CTLA4; alleviating CTLA4 immunosuppression in an organism, or Increased expression of IL-2 in T lymphocytes In another aspect, there is provided a bispecific antibody or conjugate according to any embodiment of the invention for use in

[0065] Antibody drugs, especially monoclonal antibodies (mAbs), have achieved good efficacy in treating various diseases. The traditional experimental method for obtaining these therapeutic antibodies is to immunize animals with an antigen and obtain antibodies that target the antigen in the immunized animals, or to improve those antibodies with lower affinity for the antigen by affinity maturation.

[0066] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, or complementarity-determining regions (CDRs) (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3; as defined by Kabat et al.; see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda, Md.).

[0067] The amino acid sequences of the CDR regions of the monoclonal antibodies in (1) to (13) below were analyzed by technical means well known to those skilled in the art, for example, using the VBASE2 database, and the results are as follows:

[0068] (1)14C12 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:16. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 27) HCDR2: ISGGGRYT (SEQ ID NO: 28) HCDR3: ANRYGEAWFAY (SEQ ID NO: 29) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO: 30) LCDR2:RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (SEQ ID NO: 32)

[0069] (2)14C12H1L1 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:20. The amino acid sequences of the three CDR regions of the heavy chain variable region are the same as those of 14C12. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 14C12.

[0070] (3) 4G10 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:4. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYSFTGYT (SEQ ID NO: 33) HCDR2: INPYNNIT (SEQ ID NO: 34) HCDR3: ARLDYRSY (SEQ ID NO: 35) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: TGAVTTSNF (SEQ ID NO: 36) LCDR2:GTN (SEQ ID NO: 37) LCDR3:ALWYSNHWV (SEQ ID NO: 38)

[0071] (4)4G10H1L1 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8; The amino acid sequences of the three CDR regions of the heavy chain variable region are the same as those of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 4G10.

[0072] (5)4G10H3L3 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:12. The amino acid sequences of the three CDR regions of the heavy chain variable region are the same as those of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 4G10.

[0073] (6) BiAb001(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 27) HCDR2: ISGGGRYT (SEQ ID NO: 28) HCDR3: ANRYGEAWFAY (SEQ ID NO: 29) HCDR4: GYSFTGYT (SEQ ID NO: 33) HCDR5: INPYNNIT (SEQ ID NO: 34) HCDR6: ARLDYRSY (SEQ ID NO: 35) HCDR7: TGAVTTSNF (SEQ ID NO: 36) HCDR8:GTN (SEQ ID NO: 37) HCDR9: ALWYSNHWV (SEQ ID NO: 38) The amino acid sequences of the three CDR regions associated with the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO: 30) LCDR2:RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (SEQ ID NO: 32)

[0074] (7)BiAb002(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as BiAb001(M).

[0075] (8)BiAb003(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as BiAb001(M).

[0076] (9)BiAb004(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as BiAb001(M).

[0077] For the antibody BiAb004(hG1™) of the present invention, amino acid mutations are introduced in the non-variable region of BiAb004(M). According to the EU numbering system, the amino acid mutations are introduced at positions 234, 235, and 237:

[0078] BiAb004 (hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the hinge region of the heavy chain.

[0079] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Additionally, the experimental procedures of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures widely used in the corresponding fields. In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0080] As used herein, when referring to the amino acid sequence of CTLA4 protein (cytotoxic T-lymphocyte antigen 4), it includes the full-length CTLA4 protein, or the extracellular fragment of CTLA4, CTLA4ECD, or a fragment containing CTLA4ECD; it also includes fusion proteins of CTLA4ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally or artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in the present invention, the term "CTLA4 protein" should include all such sequences, including natural or artificial variants thereof. Additionally, when a sequence fragment of the CTLA4 protein is described, it also includes the corresponding sequence fragment in the natural or artificial variant.

[0081] As used herein, reference to the amino acid sequence of the PD-1 protein (NCBI GenBank: NM_005018) includes the full-length PD-1 protein, the extracellular fragment of PD-1, PD-1ECD, or fragments containing PD-1ECD; fusion proteins of PD-1ECD, such as fragments fused to the Fc protein fragments (mFc or hFc) of mouse or human IgG, are also included. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally or artificially introduced into the amino acid sequence of the PD-1 protein without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" should include all such sequences, including natural or artificial variants thereof. Additionally, when a sequence fragment of the PD-1 protein is described, it also includes the corresponding sequence fragment in the natural or artificial variant.

[0082] As used herein, unless otherwise specified, B7 refers to B7-1 and / or B7-2, the specific sequences of which are known in the prior art, and reference may be made to sequences disclosed in existing literature or GenBank, e.g., B7-1 (CD80, NCBI Gene ID: 941); B7-2 (CD86, NCBI Gene ID: 942).

[0083] As used herein, EC 50 The term refers to the concentration for 50% of the maximal effect, ie the concentration capable of producing 50% of the maximal effect.

[0084] As used herein, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. In a general sense, a heavy chain can be understood as the polypeptide chain having the larger molecular weight in an antibody, and a light chain refers to the polypeptide chain having the smaller molecular weight in an antibody. Light chains are classified as kappa and lambda light chains. Heavy chains are generally classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also include a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 , and C H3 Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H and V L The regions can be further divided into highly variable regions (called complementarity determining regions (CDRs)) interspersed with conserved regions called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) each form an antibody-binding site. The assignment of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol., 196:901-917, or Chothia et al. (1989) Nature, 342:878-883. In particular, the heavy chain may also contain more than three CDRs, for example, six, nine, or twelve CDRs. For example, in the bispecific antibody of the present invention, the heavy chain may be an scFv in which the C-terminus of the heavy chain of an IgG antibody is linked to another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be of different isotypes, for example IgG (eg, subtypes IgG1, IgG2, IgG3 or IgG4), IgA1, IgA2, IgD, IgE or IgM.

[0085] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., DNA recombination, or enzymatic or chemical cleavage), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as intact antibodies.

[0086] As used herein, unless the context clearly defines otherwise, when the term "antibody" is used it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0087] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or fragment thereof derived from a population of highly homologous antibodies, i.e., derived from a population of identical antibody molecules except for naturally occurring mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. In contrast to monoclonal antibodies, polyclonal antibodies generally comprise at least two or more different antibodies that generally identify different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology first described by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using DNA recombinant technology (see, e.g., U.S. Pat. No. 4,816,567).

[0088] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. Additionally, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced with corresponding amino acid residues of a non-human antibody or with amino acid residues of other antibodies to further improve or optimize antibody performance. For more details regarding humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000).

[0089] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids, carbohydrates, or sugar side chains, and usually have distinctive three-dimensional structural and charge characteristics. For example, an epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation that can be "linear" or "conformational." See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are located across amino acid residues of the protein that are separated from one another.

[0090] As used herein, the term "isolated" refers to something obtained by artificial means from a natural state. When a particular "isolated" substance or component is found in nature, it may be true that the change occurred in its natural environment, or that it has been isolated from its natural environment, or both. For example, if a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular living animal, and the same polynucleotide or polypeptide has a high degree of purity and is isolated from such a natural state, it is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0091] As used herein, the term "E. coli expression system" refers to an expression system consisting of an E. coli (E. coli) strain and a vector, where the E. coli (E. coli) strain is derived from commercially available strains, such as, but not limited to, GI698, ER2566, BL21(DE3), B834(DE3), and BLR(DE3).

[0092] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a 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 the vector 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); phages, such as lambda 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 (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may further contain a replication origin.

[0093] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK293 cell, or human cell.

[0094] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen that it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) binds to an antigen when the antibody binds to an antigen within about 10 -5 Lower than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Lower or lower affinity (K D ) refers to binding to an antigen. In some embodiments of the invention, the term "targeting" refers to specific binding.

[0095] As used herein, "K D The term "antibody-antigen dissociation constant" refers to the dissociation equilibrium constant for a specific 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. Generally, antibodies have a dissociation equilibrium constant of about 10, as measured, for example, by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio Octet system. -5 Lower than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 A dissociation equilibrium constant (K D) binds to the antigen.

[0096] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably; and the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also, herein, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0097] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania, Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0098] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to an organism together with or prior to an antigen, can enhance or alter the immune response of the organism to the antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0099] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount (e.g., for a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., a tumor) is an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., a tumor); a therapeutically effective amount is an amount sufficient to cure or at least partially stop the disease and its complications in a patient suffering from the disease. Determining such an effective amount is undoubtedly within the capabilities of one skilled in the art. For example, an amount effective for therapeutic purposes depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, e.g., age, weight, and sex, the route of administration, and other concurrently administered treatments, etc.

[0100] A "recurrent" cancer is one that recurs at the original site or a distant site after responding to previous treatment (e.g., surgery). A "locally recurrent" cancer is one that occurs after treatment in the same site as the previously treated cancer.

[0101] "Metastatic" cancer refers to cancer that has spread from one part of the body (e.g., the lungs) to another.

[0102] As used herein, the term "completely eliminated" refers to the absence of a binding signal or an extremely weak binding signal as detected by existing instrumentation (e.g., the Fortebio Octet system). In one embodiment of the present invention, the absence of a binding signal or an extremely weak binding signal refers to a binding signal (i.e., response) of less than 0.1 nm.

[0103] In the present invention, the terms "first" (e.g., first protein functional region, first linker fragment, or first product) and "second" (e.g., second protein functional region, second linker fragment, or second product) are used for distinction or clarity of expression and do not have a typical sequential meaning, unless otherwise specified.

[0104] In the present invention, "about" or "approximately" means that the defined value or physical quantity may be increased or decreased within a range of 10%, 20%, or 30%, unless otherwise specified. For example, about 100 minutes or approximately 100 minutes may be 90 to 110 minutes, 80 to 120 minutes, or 70 to 130 minutes.

[0105] Advantages of this invention: The present invention achieves one or more of the following technical effects (1) to (3): (1) The inventors' modification of the Fc fragment of the antibody of the present invention completely eliminates the binding activity of BiAb004 (hG1™) to FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIIa_F158, thereby eliminating ADCC activity and / or ADCP activity. (2) The inventors' modification of the Fc fragment of the antibody of the present invention completely eliminates the binding activity to complement C1q, thereby eliminating CDC activity. (3) The antibodies of the present invention have the potential for use in the preparation of medicaments for preventing and treating tumors. [Brief explanation of the drawings]

[0106] [Figure 1] Affinity constant assay of BiAb004 (hG1™) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 2] Affinity constant assay of BiAb004 (hG1WT) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 3] Affinity constant assay of BiAb004 (hG1™) for FcγRIIIa_V158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 4] Affinity constant assay of BiAb004 (hG1WT) for FcγRIIIa_V158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 5] Affinity constant assay of BiAb004 (hG1™) for FcγRIIIa_F158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 6] Affinity constant assay of BiAb004 (hG1WT) for FcγRIIIa_F158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 7] Affinity constant assay of BiAb004 (hG1™) for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 8]Affinity constant assay of BiAb004 (hG1WT) for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 9] Affinity constant assay of BiAb004 (hG1™) for FcγRIIa_R131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 10] Affinity constant assay of BiAb004 (hG1WT) for FcγRIIa_R131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 11] Affinity constant assay of BiAb004 (hG1™) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 12] Affinity constant assay of BiAb004 (hG1WT) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 13] Affinity constant assay of BiAb004 (hG1™) for C1q. Antibody concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 14] Affinity constant assay of BiAb004 (hG1WT) for C1q. The antigen concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 15] Affinity constant assay of 5C10H2L2-IgG1mt for C1q. The antigen concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 16]ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) against 293T-CTLA4-PD1 cells expressing CTLA-4 and PD-1 antigens. [Figure 17] Inhibition of subcutaneous human lung cancer HCC827 cell tumor xenografts by BiAb004 (hG1™) in combination with lenvatinib. [Figure 18] Inhibition of subcutaneous colon cancer MC38-hPDL1 / hCD73 tumor xenografts by BiAb004 (hG1™) in C57BL / 6-hPD1 / hPDL1 / hCD73 mice. [Figure 19] Antibody-mediated phagocytic activity of BiAb004 (hG1™) against CHO-K1-PD1. [Figure 20] BiAb004 (hG1™) significantly enhanced immune cell responses against human gastric cancer KATO III cells. [Figure 21] BiAb004 (hG1™) significantly enhanced immune cell responses against human cervical cancer HeLa cells. [Figure 22] BiAb004 (hG1™) significantly enhanced immune cell responses against human T-cell lymphoma Jurkat cells. [Figure 23] Significantly enhanced immune response of immune cells against human nasopharyngeal carcinoma CNE-2Z cells by BiAb004 (hG1™). [Figure 24] Significantly enhanced immune response of immune cells against human breast cancer MDA-MB-231 cells by BiAb004 (hG1™). [Figure 25] Significantly enhanced immune response of immune cells against human mesothelioma NCI-H2452 cells by BiAb004 (hG1™). [Figure 26] Significantly enhanced immune cell responses against human non-small cell lung cancer (human lung adenocarcinoma) A549 cells by BiAb004 (hG1™) in combination with anlotinib. [Figure 27]Significantly enhanced immune response of immune cells against human small cell lung cancer NCI-H446 cells by BiAb004 (hG1™) in combination with anlotinib. [Figure 28] Significantly enhanced immune response of immune cells against human squamous cell lung cancer NCI-H226 cells by BiAb004 (hG1™) in combination with anlotinib. [Figure 29] Significantly enhanced immune response of immune cells against MSI-H / dMMR phenotype human colorectal cancer SW48 cells by BiAb004 (hG1™). [Figure 30] Significantly enhanced immune response of immune cells against non-MSI-H / dMMR phenotype human colorectal cancer SW837 cells by BiAb004 (hG1™). DETAILED DESCRIPTION OF THE INVENTION

[0107] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no techniques or conditions are specified, the examples are carried out according to the techniques or conditions described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., Third Edition, Science Press) or according to the product manual. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that are commercially available.

[0108] In the following examples of the present invention: BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center. Human peripheral blood mononuclear cells were isolated and prepared at Akeso Biopharma, Inc. with informed consent of the donors. Raji-PDL1 is a cell line expressing human PD-L1 that was constructed by Akeso Biopharma via transfection based on the human B cell line Raji. Ficoll-Paque TM PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare. Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd. RPMI 1640 medium, DMEM medium, trypsin-EDTA (0.25%), phenol red, and blastidin were all purchased from Gibco. Staphylococcus aureus enterotoxin B (SEB) was purchased from Dianotech. FBS was purchased from Excell bio. Mitomycin C (MMC) was purchased from Stressmarq. The sequence of the isotype control, human anti-hen egg lysozyme IgG (anti-HEL antibody, or human IgG abbreviated as hIgG), was derived from the variable region sequence of Fab F10.6.6 in the study reported by Acierno et al. entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007; 374(1):130-146). The anlotinib used in the examples is the hydrochloride salt of anlotinib under the trade name Fukewei® and the generic name anlodinib hydrochloride, and was purchased from CTTQ Pharma. The lenvatinib used in the examples was lenvatinib mesylate under the trade name Lenvima® and was purchased from Eisai (China).

[0109] Preparation Example 1: Sequence design of anti-CTLA4 antibody The amino acid sequences and coding nucleotide sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1 and 4G10H3L3, respectively, in Chinese Patent Publication No. 106967172(A).

[0110] (1) 4G10 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region: (372 bp) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAG TTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO: 1) Encoded amino acid sequence: (124 aa) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2) Nucleotide sequence of the light chain variable region: (378bp) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTC TCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (SEQ ID NO: 3) Encoded amino acid sequence: (126 aa) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4)

[0111] (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H1L1 Nucleotide sequence of the heavy chain variable region (4G10H1V): (345bp) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACCAA CTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT (SEQ ID NO: 5) Encoded amino acid sequence: (115 aa) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 6) Nucleotide sequence of the light chain variable region (4G10L1V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAATCG GGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG (SEQ ID NO: 7) Encoded amino acid sequence: (109 aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 8)

[0112] (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H3L3 Nucleotide sequence of the heavy chain variable region (4G10H3V): (345bp) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAA CTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (SEQ ID NO: 9) Encoded amino acid sequence (4G10H3V): (115 aa) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 10) Nucleotide sequence of the light chain variable region (4G10L3V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAACAA GGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (SEQ ID NO: 11) Encoded amino acid sequence (4G10L3V): (109aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 12)

[0113] Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences and coding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172(A).

[0114] (1) Heavy and light chain variable region sequences of 14C12 Nucleotide sequence of the heavy chain variable region: (354 bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATC CTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 13) Encoded amino acid sequence: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 14) Nucleotide sequence of the light chain variable region: (321 bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 15) Encoded amino acid sequence: (107aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 16)

[0115] (2) Heavy and light chain variable regions and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Nucleotide sequence of the heavy chain variable region: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 17) Encoded amino acid sequence: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18) Nucleotide sequence of the light chain variable region: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 19) Encoded amino acid sequence: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 20) DNA sequence of 14C12H1L1 heavy chain (14C12H1): (1344 bp) Encoded amino acid sequence: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) DNA sequence of 14C12H1L1 light chain (14C12L1): (642bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 23) Encoded amino acid sequence: (214aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24)

[0116] Preparation Example 3: Sequence design of bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) The structural pattern of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of one IgG antibody is separately linked to the scFv fragment of another antibody via a linker fragment. The components for the design of the heavy and light chains are shown in Table A below. [Table 1] In Table A above: The amino acid sequence of linker 1 is (GGGGS)3 (SEQ ID NO:25), and The amino acid sequence of linker 2 is (GGGGS)4 (SEQ ID NO: 26).

[0117] In Table A above, the scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M) and 4G10L3V(M) of the BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) antibodies contain mutations in individual amino acids of the framework regions based on 4G10H1V, 4G10L1V, 4G10H3V and 4G10L3V, respectively, which effectively optimized the structure of the antibodies and improved their efficacy.

[0118] (1) 4G10H1V(M): (115 aa, mutations are underlined in the amino acid sequence based on 4G10H1V) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 41) (2) 4G10L1V(M): (110 aa, the positions of mutations are underlined in the amino acid sequence based on 4G10L1V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 42) (3) 4G10H3V(M): (115 aa, mutations are underlined in the amino acid sequence based on 4G10H3V) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 43) (4) 4G10L3V(M): (110 aa, the positions of mutations are underlined in the amino acid sequence based on 4G10L3V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 44)

[0119] To distinguish it from the mutated antibody, BiAb004(M) is also referred to as BiAb004(hG1WT) in this example. The above-mentioned BiAb004(M) is a "wild-type" antibody that contains an Ig gamma-1 chain C region (accession number P01857) as the heavy chain constant region and an Ig kappa chain C region (accession number P01834) as the light chain constant region.

[0120] Preparation Example 4: Design of non-variable region amino acid mutations based on humanized bifunctional antibody BiAb004 Based on BiAb004(hG1WT) obtained in Preparation Example 3, BiAb004(hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 in the heavy chain (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A).

[0121] DNA sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1™): (1344 bp, positions of mutations are underlined) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCT ACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCT AGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGC AAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCC CCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA(SEQ ID NO: 39)

[0122] Amino acid sequence of the heavy chain of the immunoglobulin portion in BiAb004 (hG1TM): (448 aa, positions of mutations are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0123] BiAb004(hG1TM) and BiAb004(hG1WT) share the same DNA sequence and the same encoded amino acid sequence for the light chain. The unique sequence is shown in Preparation 3.

[0124] Experimental Example 1: Affinity assay of BiAb004(hG1WT) and BiAb004(hG1TM) for the receptor FcγRI The Fc receptor FcγRI, also known as CD64, can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The binding ability of a therapeutic monoclonal antibody to an Fc receptor affects the safety and efficacy of the antibody.

[0125] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRI were measured in this experiment using the Fortebio Octet system to assess the potential ADCC and ADCP activities of the antibodies.

[0126] The method for determining the affinity constant of an antibody for FcγRI using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. A 1 μg / mL FcγRI solution (Sinobio) was added to the HIS1K sensor, and FcγRI was immobilized on the sensor surface for 50 s. Both the association and dissociation constants of the antibody for FcγRI were determined in buffer using antibody concentrations ranging from 3.12 to 50 nM (serial 2-fold dilutions). The antigen-immobilized sensor was equilibrated in buffer for 60 s, and then binding of the antibody to the immobilized FcγRI on the sensor was determined for 120 s; dissociation of FcγRI from the antibody was determined for 120 s. The temperature was 30°C, and the frequency was 0.3 Hz. The data were fitted and analyzed using a 1:1 model to obtain the affinity constant of the antibody for FcγRI.

[0127] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRI are shown in Table 1 below and in FIGS. [Table 2] The results showed that BiAb004(hG1WT) bound to FcγRI with an affinity constant of 5.92E-09M; however, no corresponding data was obtained for BiAb004(hG1TM), and either no or very weak binding signal to FcγRI was detected, indicating no binding to FcγRI.

[0128] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRI is effectively eliminated compared to BiAb004(hG1WT).

[0129] Experimental Example 2: Affinity constant assay of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIIa_V158 The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0130] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_V158 were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0131] The method for determining the affinity constant of an antibody for FcγRIIIa_V158 using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on a HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and the binding of antibodies at concentrations ranging from 31.25 to 500 nM (serial two-fold dilutions) to the immobilized FcγRIIIa_V158 on the sensor was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was regenerated four times in 10 mM glycine (pH 1.5), each time for 5 s. The temperature was 30 °C, and the frequency was 0.3 Hz. The data were analyzed by fitting to a 1:1 model to obtain the affinity constant.

[0132] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIIa_V158 are shown in Table 2 below and in FIGS. 3 and 4. [Table 3] The results showed that BiAb004(hG1WT) bound to FcγRIIIa_V158 with an affinity constant of 1.77E-07M; however, for BiAb004(hG1TM), no corresponding data were obtained and either no or very weak binding signal to FcγRIIIa_V158 was detected, indicating no binding to FcγRIIIa_V158.

[0133] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_V158 is effectively eliminated compared to BiAb004(hG1WT).

[0134] Experimental Example 3: Affinity constant assay of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIIa_F158 The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0135] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0136] The method for determining the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and binding of the immobilized FcγRIIIa_F158 to antibodies at concentrations ranging from 31.25 to 500 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was regenerated four times in 10 mM glycine (pH 1.5), for 5 s each. The temperature was 30 °C, and the frequency was 0.3 Hz. The data were analyzed by fitting to a 1:1 model to obtain affinity constants.

[0137] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIIa_F158 are shown in Table 3 below and in FIGS. 5 and 6. [Table 4] The results showed that BiAb004(hG1WT) bound to FcγRIIIa_F158 with an affinity constant of 2.21E-07M; however, for BiAb004(hG1TM), no corresponding data were obtained and either no or very weak binding signal to FcγRIIIa_F158 was detected, indicating no binding to FcγRIIIa_F158.

[0138] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_F158 is effectively eliminated compared to BiAb004(hG1WT).

[0139] Experimental Example 4: Affinity constant assay of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIa_H131 The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0140] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_H131 were measured in this experiment using the Fortebio Octet system to assess the ADCC activity of the antibodies.

[0141] The method for determining the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_H131 using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated with PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, for 300 s with blocking. Binding of the antibody to the immobilized FcγRIIa_H131 on the sensor was determined for 60 s at concentrations ranging from 12.5 to 200 nM (serial two-fold dilutions). The antibody was allowed to dissociate in the buffer for 60 s. The sensor was regenerated in 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data were analyzed by fitting to a 1:1 model to obtain affinity constants.

[0142] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIa_H131 are shown in Table 4 below and in FIGS. [Table 5] The results showed that BiAb004(hG1WT) bound to FcγRIIa_H131 with an affinity constant of 2.22E-08M; however, for BiAb004(hG1TM), no corresponding data were obtained and either no or very weak binding signal to FcγRIIa_H131 was detected, indicating no binding to FcγRIIa_H131.

[0143] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIa_H131 is effectively eliminated compared to BiAb004(hG1WT).

[0144] Experimental Example 5: Affinity constant assay of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIa_R131 The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) binds the Fc fragment of IgG antibodies and can mediate ADCC effects.

[0145] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_R131 were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0146] The method for determining the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated with PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, for 300 s with blocking. Binding of the antibody to the immobilized FcγRIIa_R131 on the sensor was determined for 60 s at concentrations ranging from 12.5 to 200 nM (serial two-fold dilutions). The antibody was allowed to dissociate in the buffer for 60 s. The sensor was regenerated in 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data were analyzed by fitting to a 1:1 model to obtain affinity constants.

[0147] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIa_R131 are shown in Table 5 below and in FIGS. 9 and 10. [Table 6] The results showed that both BiAb004(hG1WT) and BiAb004(hG1TM) bound to FcγRIIa_R131 with affinity constants of 1.43E-08 M and 4.20E-08 M, respectively.

[0148] The results suggest that both BiAb004(hG1WT) and BiAb004(hG1TM) have binding activity to FcγRIIa_R131. However, the results in Figures 9 and 10 show that BiAb004(hG1WT) has a stronger binding signal and therefore a higher affinity than BiAb004(hG1TM).

[0149] Experimental Example 6: Affinity constant assay of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIb The Fc receptor FcγRIIb (also known as CD32b) binds to the Fc fragment of IgG antibodies and can downregulate immune cell function, inhibit immune cell activation and proliferation, and inhibit cytokine secretion.

[0150] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb were measured in this experiment using the Fortebio Octet system to evaluate the binding ability of BiAb004(hG1WT) and BiAb004(hG1TM) to Fc receptors.

[0151] The method for determining the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIb was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated with PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, for 300 s with blocking, and the binding of antibody to the immobilized FcγRIIb on the sensor was determined for 60 s at concentrations ranging from 12.5 to 200 nM (serial two-fold dilutions). The antibody was allowed to dissociate in the buffer for 60 s. The sensor was regenerated in 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data were analyzed by fitting to a 1:1 model to obtain affinity constants.

[0152] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIb are shown in Table 6 below and in FIGS. 11 and 12. [Table 7] The results showed that BiAb004(hG1WT) bound to FcγRIIb with an affinity constant of 5.61E-08M; however, no corresponding data was obtained for BiAb004(hG1TM), and either no or very weak binding signal to FcγRIIb was detected, indicating no binding to FcγRIIb. The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIb is effectively eliminated compared to BiAb004(hG1WT).

[0153] Experimental Example 7: Affinity assay of BiAb004(hG1WT) and BiAb004(hG1TM) for C1q Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate the CDC effect. Binding of therapeutic monoclonal antibodies to C1q affects the safety and efficacy of the antibody.

[0154] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for C1q were measured in this experiment using the Fortebio Octet system to evaluate the CDC activity of the antibodies. In this example, the anti-PDL1 antibody 5C10H2L2-IgG1mt was used as a control, the preparation of which is described in PCT Publication WO 2017148424(A1).

[0155] The method for determining the affinity constant of an antibody for C1q using the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on a FAB2G sensor at an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s for blocking, and binding of the immobilized antibody on the sensor to the antigen C1q at concentrations ranging from 1.25 to 20 nM (serial two-fold dilutions) was determined for 60 s. The antigen and antibody were allowed to dissociate in the buffer for 60 s. The sensor was regenerated four times in 10 mM glycine (pH 1.7), each for 5 s. The sample plate was shaken at 1000 rpm, 30 °C, and 0.6 Hz. The data were analyzed by fitting to a 1:1 model to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0156] The results of the affinity constant assay of BiAb004(hG1TM), BiAb004(hG1WT) and 5C10H2L2-IgG1mt for C1q are shown in Table 7 below and in Figures 13, 14 and 15. [Table 8] The results showed that BiAb004(hG1WT) bound to C1q with an affinity constant of 2.53E-09M; however, for BiAb004(hG1TM), no corresponding data were obtained and either no binding signal to C1q or a very weak signal was detected, indicating no binding to C1q.

[0157] The results suggest that the binding activity of BiAb004(hG1TM) to C1q is effectively eliminated compared to BiAb004(hG1WT).

[0158] Experimental Example 8: ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) in 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens The ADCC effect refers to the direct killing of target cells by effector immune cells with killing activity, which recognize the Fc fragment of an antibody bound to an antigen on the target cell through the Fc receptor (FcR) expressed on their surface. To test the ADCC effect of the anti-CTLA4 / anti-PD-1 bifunctional antibodies BiAb004(hG1WT) and BiAb004(hG1TM), the inventors established a co-culture system of 293T-CTLA4-PD1 cells expressing PD-1 and CTLA4 antigens and primary PBMCs to test the ADCC activity of the antibodies.

[0159] The ADCC activity assay method for BiAb004(hG1WT) and BiAb004(hG1TM) in 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens is as follows: Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation instructions. The isolated PBMCs were resuspended in RPMI-1640 complete medium, stained with AO / PI, counted, and frozen. One day before the experiment, the PBMCs were thawed, counted, and viability determined. The cells were incubated overnight in a carbon dioxide incubator at 5% CO2 and 37°C. On the day of the experiment, 293T-CTLA4-PD1 cells and PBMCs were collected, centrifuged at 800 rpm or 1200 rpm for 5 minutes, resuspended in RPMI-1640 (containing 1% FBS; hereafter referred to as medium), and washed twice. The cells were counted and viability determined. The cell density was adjusted to an appropriate range using medium. 100 μL of 293T-CTLA4-PD1 cells were added to a 96-well plate at a density of 3.0E+04 cells / well according to the experimental design. 50 μL of antibody was added, and the cells were preincubated at room temperature for 1 h; after 1 h, 50 μL of PBMCs were added at 9.0E+05 cells / well, the mixture was mixed well, and the mixture was incubated at 37°C in a carbon dioxide incubator with 5% CO2 for 4 h. The cells were centrifuged at 250 × g for 5 min. 100 μL of the supernatant was transferred to a new 96-well flat-bottom microplate (without pipetting the cell pellet). 100 μL of freshly prepared reaction solution according to the Cytotoxicity Detection Kit instructions was added to each well. The cells were incubated at room temperature in the dark for 30 min. OD values ​​at 490 nm and 650 nm were measured. The ADCC percentage for each group was calculated according to the formula: ADCC (%) = (treated group - negative control group) / (maximum LDH release in target cells - spontaneous LDH release in target cells) × 100%.

[0160] The ADCC activity of BiAb004(hG1WT) and BiAb004(hG1TM) in 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens was expressed as ADCC percentage, and the results are shown in FIG.

[0161] The results showed that in a mixed culture system of PBMC and 293T-CTLA4-PD1, the ADCC percentage induced by BiAb004(hG1™) was significantly lower than that induced by the same level of BiAb004(hG1WT).

[0162] The results suggest that BiAb004(hG1™) has no ADCC activity.

[0163] Experimental Example 9: Growth inhibition of colorectal cancer MC38 cell xenografts in mice by BiAb004 (hG1™) in combination with lenvatinib The murine MC38 cell line is a murine colorectal cancer cell line. The MC38 cell line has been demonstrated to be a useful model for studying human MSI-H / dMMR tumors (Efremova M et al., Nat Commun., 2018; 9(1):32).

[0164] MC-38 cells (purchased from Shanghai Ruilu Biotech Co., Ltd.) were harvested, adjusted to a density of 5 million cells / mL, and then implanted subcutaneously into the right flank of PD-1 transgenic mice (purchased from Shanghai Model Organisms Center, Inc.) at a volume of 200 μL / mouse. Twelve mice were implanted, each containing 1 × 10 6 The tumor volume was approximately 60-150 mm 3 When the tumor volume reached 94 mm, the mice were cultured. 3 The mice were divided into two groups, each containing 6 mice, a model group and a BiAb004(hG1™) / lenvatinib treatment group, according to the tumor size (having similar average volumes of 1000-120 ... 2Tumor volume was calculated according to (a is the longest diameter of the tumor, b is the shortest diameter of the tumor, and TV is the volume of the tumor). The specific protocol is shown in Table 8. [Table 9] The sequence of the isotype control in this experiment, human anti-hen egg lysozyme IgG (anti-HEL antibody, or human IgG abbreviated as hIgG), was derived from the variable region sequence of Fab F10.6.6 in the study reported by Acierno et al. titled "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).

[0165] The experimental results are shown in FIG.

[0166] The results showed that BiAb004(hG1™) in combination with lenvatinib significantly inhibited the growth of MC38 tumors; BiAb004(hG1™) in combination with lenvatinib demonstrated significant efficacy against rectal and / or colon cancer with MSI-H / dMMR phenotype.

[0167] Experimental Example 10: Pharmacodynamic evaluation of BiAb004 (hG1™) in C57BL / 6-hPD1 / hPDL1 / hCD73 mouse colon cancer MC38-hPDL1 / hCD73 subcutaneous xenograft tumor models Female C57BL / 6-hPD1 / hPDL1 / hCD73 mice (purchased from Nanjing GemPharmatech Co., Ltd.) were divided into eight groups and colon cancer MC38-hPDL1 / hCD73 cells (purchased from Nanjing GemPharmatech Co., Ltd.) (2 × 10 6The day of implantation was defined as D0. The dosing volume was adjusted according to body weight, to 10 μL per gram of mouse body weight. Isotype control antibody (prepared and supplied by the same supplier as in Example 9) or BiAb004 (hG1™) was administered intraperitoneally twice weekly for 3 weeks for a total of 6 doses. Tumors were measured continuously throughout the experiment and calculated using the following formula: tumor volume (mm 3 ) = (tumor length × (tumor width) 2 The volume was calculated as ) / 2.

[0168] The results are shown in Figure 18 and Table 9 below. [Table 10] The results showed that the increase in tumor volume in the BiAb004(hG1™) group was inhibited compared to the isotype control antibody group, indicating that BiAb004(hG1™) significantly inhibited the proliferation of mouse MC38 cells and could effectively treat colon and / or rectal cancer.

[0169] Experimental Example 11: Study of antibody-mediated phagocytic activity of BiAb004(hG1WT) and BiAb004(hG1TM) in CHO-K1-PD1 Zhang et al. (Zhang T et al, Cancer Immunol Immunother., 2018; 67(7):1079-1090.) and Dahan et al. (Dahan R et al., Cancer cell, 2015, 28(3):285-95.) reported that binding of Fc fragments of antibodies targeting immune checkpoints, such as PD-1 and CTLA-4, to Fc receptors negatively affects antibody-mediated anticancer activity, possibly due to Fc-dependent effector function-induced immune cell damage, an important mechanism by which antibody-dependent cellular phagocytosis (ADCP) leads to immune cell damage.

[0170] To test the ADCP activity of BiAb004 (hG1™), mouse macrophages were used as effector cells, and a cell line overexpressing the corresponding antigen was used as target cells. Cell-mediated ADCP effects were examined. Femoral bone marrow from C57BL / 6 mice (purchased from the Guangdong Medical Laboratory Animal Center) was first aseptically collected and lysed with red blood cell lysis buffer on ice for 5 minutes. Lysis was terminated with DMEM complete medium (containing 10% FBS), and the lysate was centrifuged at 1000 rpm and washed twice. The cell pellet was resuspended in 10 mL of DMEM complete medium, and macrophage colony-stimulating factor (M-CSF) was added at a working concentration of 100 ng / mL. For induction, the cells were cultured for 7 days at 37°C and 5% CO2 in a cell culture chamber. Half of the medium was replaced on days 3 and 5, and M-CSF was added. Cell induction was completed on day 7. The cells were digested with 0.25% trypsin. Macrophages were collected and centrifuged at 170 x g for 5 minutes. The supernatant was discarded, and the cells were suspended in DMEM complete medium and counted. The cells were adjusted to the appropriate density and packed into sterile EP tubes for further use.

[0171] Logarithmic-phase CHO-K1-CTLA4-PD1 cells (a CHO-K1 cell-based cell line overexpressing both human CTLA4 and PD1 antigens, constructed by Akeso Biopharma, Inc.) were harvested, centrifuged at 170 × g for 5 minutes, washed with PBS, resuspended, and counted to determine viability. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM with PBS, and the cells were resuspended (staining density: 10 million cells / mL). The cells were incubated in a cell incubator for 20 minutes. Staining was stopped by adding 6 mL of DMEM complete medium. The cells were centrifuged at 170 × g for 5 minutes, and the supernatant was discarded. 1 mL of DMEM complete medium was added for resuspension. The cells were incubated in an incubator for 10 minutes and adjusted to the experimental density. The cells were coded as CHO-K1-PD1-CTLA4-CFSE. Test antibodies were diluted in DMEM complete medium. Isotype control antibody anti-HEL antibody and medium were used as isotype and blank controls. According to the study design, diluted antibody and CHO-K1-PD1-CTLA4-CFSE cells were added to 1.5 mL EP tubes containing macrophages (final volume was 100 μL, effector-to-target ratio was 50,000:150,000, and working antibody concentrations were 50, 5, and 0.5 nM). The mixture was resuspended, mixed uniformly, and incubated for 2 h at 37°C in an incubator. 800 μL of PBS containing 1% bovine serum albumin (BSA) was added to each tube at room temperature. The mixture was centrifuged at 1200 × g for 5 minutes, and the supernatant was discarded. The cells were washed once with 800 μL of 1% PBSA. APC anti-mouse / human CD11b antibody (Biolegend, product number: 101212) was diluted 400-fold with 1% PBSA and added to the corresponding samples at 100 μL per sample. The mixture was mixed well, incubated on ice for 30 minutes, washed once with 800 μL of 1% PBSA, centrifuged at 1200 × g for 5 minutes, and the supernatant was discarded. 200 μL of 1% PBSA was added to each tube to resuspend the cells. The cells were transferred to a loading tube and analyzed using a BD FACSCalibur flow cytometer. Macrophages in the system were APC+ Macrophages that were positive and involved in phagocytosis were APC and CFSE double positive. The phagocytosis rate was determined as the ratio of the number of double positive cells to the number of APC positive cells, and antibody-mediated ADCP activity was evaluated.

[0172] The ADCP activity of each group, expressed as P%, was calculated as follows:

number

[0173] In an antibody validation system for mediation of phagocytic activity, the results showed that the phagocytic activity of CHO-K1-PD1-CTLA4 cells by macrophages mediated by BiAb004(hG1™) was not significantly different from that of the isotype control antibody anti-HEL antibody compared to BiAb004(hG1WT), indicating that BiAb004(hG1™) does not have ADCP activity.

[0174] The results suggest that the amino acid mutations introduced by BiAb004 (hG1™) can effectively eliminate the ADCP effect, resulting in a surprising technical effect.

[0175] Experimental Example 12: BiAb004 (hG1™) significantly enhanced immune response of immune cells against human gastric cancer KATO III cells Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and KATO III cells (purchased from the Chinese Academy of Sciences Shanghai Cell Bank) were cultured in DMEM + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL mitomycin C (MMC) for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 KATO III cells in logarithmic growth phase were harvested and plated at 5 × 10 cells / well on a 96-well plate. 4 Cells were seeded at 10 ...

[0176] The results are shown in Figure 20.

[0177] The results showed that BiAb004(hG1TM) significantly enhanced the immune response of immune cells against human gastric cancer cells KATO III, as characterized by a significantly increased secretion level of IL-2 compared with BiAb004(hG1WT), and therefore BiAb004(hG1TM) is promising for treating gastric cancer.

[0178] Experimental Example 13: BiAb004 (hG1™) significantly enhanced immune cell responses against human cervical cancer HeLa cells Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and Hela cells (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 HeLa cells in logarithmic growth phase were harvested and plated at 5 x 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0179] The results are shown in Figure 21.

[0180] The results showed that BiAb004(hG1™) significantly enhanced the immune response of immune cells against human cervical cancer Hela cells, as characterized by a significantly increased secretion level of IL-2 compared with BiAb004(hG1WT), and therefore BiAb004(hG1™) is promising for treating cervical cancer.

[0181] Experimental Example 14: Significantly enhanced immune response of immune cells against human T-cell lymphoma Jurkat cells by BiAb004 (hG1™) Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and human T-cell lymphoma Jurkat cells (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 Jurkat cells in logarithmic growth phase were harvested and plated at 1 x 10 cells / well in a 96-well plate. 5 Cells were seeded at 10 ...

[0182] The results are shown in Figure 22.

[0183] BiAb004(hG1™) significantly enhanced the immune response of immune cells against human T-cell lymphoma Jurkat cells, as characterized by significantly increased levels of IL-2 secretion compared to BiAb004(hG1WT), while eliminating ADCC and CDC (i.e., ADCP); the antibody had similar or higher pharmacological activity at doses of 1.34 nM and 20 nM compared to BiAb004(hG1WT), making it promising for treating cervical cancer.

[0184] Experimental Example 15: BiAb004 (hG1™) significantly enhanced immune cell responses against human nasopharyngeal carcinoma CNE-2Z cells Ficoll-PaqueTM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and CNE-2Z cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 CNE-2Z cells in logarithmic growth phase were harvested and plated at 3 x 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0185] The results are shown in Figure 23.

[0186] The results showed that BiAb004(hG1TM) equally and / or significantly enhanced the immune cell response against human nasopharyngeal carcinoma cells CNE-2Z, as characterized by significantly increased levels of IL-2 secretion compared to BiAb004(hG1WT), and that BiAb004(hG1TM) had a significant advantage, especially at 20 nM.

[0187] The above results showed that BiAb004(hG1™) had better or equivalent pharmacological activity compared with BiAb004(hG1WT) based on the effective elimination of ADCC, CDC and ADCP effects, indicating its potential for treating human nasopharyngeal carcinoma.

[0188] Experimental Example 16: BiAb004 (hG1™) significantly enhanced immune cell responses against human breast cancer MDA-MB-231 cells Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and MDA-MB-231 cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 MDA-MB-231 cells in logarithmic growth phase were harvested and plated at 3 × 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0189] The results are shown in Figure 24.

[0190] BiAb004(hG1TM) equally or significantly enhanced the immune cell response against human breast cancer MDA-MB-231 cells, as characterized by significantly increased levels of IL-2 secretion compared to BiAb004(hG1WT).

[0191] The above results showed that BiAb004(hG1™) has better or equivalent pharmacological activity compared with BiAb004(hG1WT) based on the effective elimination of ADCC, CDC, and ADCP effects, indicating its potential for treating human breast cancer.

[0192] Experimental Example 17: BiAb004 (hG1™) significantly enhanced immune cell responses against human mesothelioma NCI-H2452 cells Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H2452 cells (purchased from the Chinese Academy of Sciences Shanghai Cell Bank) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 NCI-H2452 cells in logarithmic growth phase were harvested and plated at 3 × 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0193] The results are shown in Figure 25.

[0194] The results showed that BiAb004(hG1™) significantly enhanced the immune response of immune cells against human mesothelioma cells NCI-H2452, as characterized by a significantly increased secretion level of IL-2 compared with BiAb004(hG1WT), and therefore BiAb004(hG1™) is promising for treating mesothelioma.

[0195] Experimental Example 18: Significantly enhanced immune response of immune cells against human non-small cell lung cancer (human lung adenocarcinoma) A549 cells by BiAb004 (hG1™) in combination with anlotinib Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and A549 cells were cultured in DMEM + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 cells per well onto a 96-well plate. 5 A549 cells in logarithmic growth phase (purchased from the Chinese Academy of Sciences Cell Bank) were harvested and seeded at 5 × 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0196] As shown in Figure 26, BiAb004(hG1™) in combination with anlotinib hydrochloride significantly enhanced the immune response of immune cells against human non-small cell lung cancer (human lung adenocarcinoma) A549 cells as characterized by significantly increased IL-2 secretion levels compared to BiAb004(hG1™) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) in combination with anlotinib hydrochloride, and therefore BiAb004(hG1™) in combination with anlotinib hydrochloride is promising for treating human non-small cell lung cancer or human lung adenocarcinoma.

[0197] Experimental Example 19: Significantly enhanced immune response of immune cells against human small cell lung cancer NCI-H446 cells by BiAb004 (hG1™) in combination with anlotinib Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H446 cells (purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 NCI-H446 cells in logarithmic growth phase (purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were harvested and seeded at 8 × 10 cells / well onto a 96-well plate. 4Cells were seeded at 10 ...

[0198] The results are shown in Figure 27.

[0199] BiAb004(hG1™) in combination with anlotinib hydrochloride significantly enhanced the immune response of immune cells against human small cell lung cancer NCI-H446 cells as characterized by significantly increased IL-2 secretion levels compared to BiAb004(hG1MT) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) in combination with anlotinib hydrochloride, and therefore BiAb004(hG1™) in combination with anlotinib hydrochloride is promising for treating human small cell lung cancer.

[0200] Experimental Example 20: Significantly enhanced immune response of immune cells against human squamous cell lung cancer NCI-H226 cells by BiAb004 (hG1™) in combination with anlotinib Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H226 cells (purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 NCI-H226 cells in logarithmic growth phase were harvested and plated at 5 × 10 cells / well onto a 96-well plate. 4 Cells were seeded at 10 ...

[0201] The results are shown in Figure 28.

[0202] BiAb004(hG1™) monotherapy more effectively and significantly enhanced the immune cell response against human squamous cell lung cancer NCI-H226 cells, as characterized by significantly increased levels of IL-2 secretion compared to BiAb004(hG1WT) monotherapy.

[0203] BiAb004(hG1TM) in combination with anlotinib hydrochloride significantly enhanced the immune cell response against human squamous cell lung cancer NCI-H226 cells compared with BiAb004(hG1WT) monotherapy and BiAb004(hG1TM) monotherapy, demonstrating pharmacological activity equivalent to that of BiAb004(hG1WT) in combination with anlotinib.

[0204] The above results showed that BiAb004(hG1™) exhibited better or equivalent pharmacological activity compared with BiAb004(hG1WT) monotherapy or BiAb004(hG1WT) in combination with anlotinib based on the effective elimination of ADCC, CDC and ADCP effects, indicating better efficacy for treating human squamous cell lung cancer.

[0205] Experimental Example 21: BiAb004 (hG1™) significantly enhanced immune cell responses against MSI-H / dMMR phenotype human colorectal cancer SW48 cells SW48 is a human colorectal cancer cell line identified with an MSI-H / dMMR phenotype (Branch P et al., (1995), Cancer Res, 55(11): 2304-2309.) We used it to detect enhanced immune cell responses against tumors with an MSI-H / dMMR phenotype induced by BiAb004 (hG1™).

[0206] In the experiment, Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and SW48 cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in DMEM + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 SW48 cells in logarithmic growth phase were harvested and plated at 2 x 10 cells / well onto a 96-well plate. 5 Cells were seeded at 10 ...

[0207] The results are shown in Figure 29.

[0208] Both BiAb004(hG1WT) and BiAb004(hG1TM) significantly enhanced immune cell responses against SW48 human colorectal cancer cells with the MSI-H / dMMR phenotype, as characterized by significantly increased levels of IL-2 secretion compared to anti-HEL antibody.

[0209] The above results showed that BiAb004(hG1™) has better or equivalent pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC, and ADCP effects, indicating its potential for treating solid tumors with MSI-H / dMMR phenotype, especially colon cancer and / or rectal cancer with MSI-H / dMMR phenotype.

[0210] Experimental Example 22: BiAb004 (hG1™) significantly enhanced immune cell responses against MSI-H / dMMR phenotype human colorectal cancer SW837 cells SW837 is a human colorectal cancer cell line with a non-MSI-H / dMMR (i.e., MSS) phenotype (Guo J et al., Cancer Res., 2011;71(8):2978-2987.), and was used in this example to detect enhanced immune cell responses against non-MSI-H / dMMR tumors by BiAb004 (hG1™).

[0211] Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the Plus Reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and SW837 cells (purchased from Shanghai Honsun Biological Technology Co., Ltd.) were cultured in 10% FBS + Leibovitz's L-15 (purchased from Gibco) complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were plated at 1 × 10 in a 96-well plate. 5 SW837 cells in logarithmic growth phase were harvested and plated at 5 x 10 cells / well onto a 96-well plate. 4Cells were seeded at 10 ...

[0212] All media in this experiment was 10% FBS + RPMI 1640.

[0213] The results are shown in Figure 30.

[0214] Both BiAb004(hG1WT) and BiAb004(hG1TM) significantly enhanced immune cell responses against non-MSI-H / dMMR human colorectal cancer cells SW837 cells compared with anti-HEL antibody. The pharmacological activity of BiAb004(hG1TM) in the high-dose group was superior to that of BiAb004(hG1WT), as characterized by significantly increased levels of IL-2 secretion.

[0215] The above results showed that BiAb004(hG1™) has better or equivalent pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC, or ADCP effects, indicating its potential for treating solid tumors with non-MSI-H / dMMR phenotype, particularly colon and / or rectal cancer with non-MSI-H / dMMR phenotype.

[0216] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to these details according to all the teachings disclosed, and all of these modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. a first protein functional region that targets PD-1; and A bispecific antibody comprising a second protein functional region that targets CTLA4, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; the amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO:40, and the amino acid sequence of the light chain of said immunoglobulin is set forth in SEQ ID NO:24; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:44; the first protein functional region is linked to the second protein functional region via a first linker fragment, and the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody via a second linker fragment, wherein the first linker fragment and the second linker fragment are the same or different; and A bispecific antibody, wherein the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

2. 2. The bispecific antibody of claim 1 , wherein the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO:25 and SEQ ID NO:

26.

3. 3. The bispecific antibody of claim 1 or 2, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

26.

4. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 3.

5. A vector comprising the isolated nucleic acid molecule of claim 4.

6. A host cell comprising the isolated nucleic acid molecule of claim 4 or the vector of claim 5.

7. A conjugate comprising the bispecific antibody of any one of claims 1 to 3 and a conjugated moiety, wherein the conjugated moiety is a detectable label.

8. A kit comprising a bispecific antibody according to any one of claims 1 to 3 or a conjugate according to claim 7, the kit further comprises a second antibody capable of specifically recognizing the bispecific antibody or an antigen-binding fragment thereof; and the second antibody further comprises a detectable label, for example, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. kit.

9. Use of a bispecific antibody according to any of claims 1 to 3 or a conjugate according to claim 7 in the preparation of a kit for detecting the presence or level of PD-1 and / or CTLA4 in a sample.

10. 8. A pharmaceutical composition comprising a bispecific antibody according to any of claims 1 to 3 or a conjugate according to claim 7, optionally said pharmaceutical composition further comprising a pharma- ceutically acceptable carrier and / or excipient.

11. 11. The pharmaceutical composition of claim 10, further comprising one or more anti-tumor chemotherapeutic agents.

12. The pharmaceutical composition of claim 11, wherein the one or more anti-tumor chemotherapeutic agents are tyrosine kinase inhibitors.

13. The pharmaceutical composition according to claim 13, wherein the tyrosine kinase inhibitor is anlotinib or a pharma- ceutically acceptable salt thereof, or lenvatinib or a pharma- ceutically acceptable salt thereof.

14. 14. The pharmaceutical composition according to any of claims 10 to 13, wherein a unit dose of the pharmaceutical composition is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg based on the mass of the bispecific antibody.

15. A combination product comprising a first product and a second product in separate packages, said first product comprising a bispecific antibody according to any one of claims 1 to 3, a conjugate according to claim 8, or a pharmaceutical composition according to any one of claims 10 to 14; the second product comprises one or more anti-tumor chemotherapeutic agents; Combination products.

16. 16. The combination product of claim 15, wherein the unit dose of the first product is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg based on the mass of the bispecific antibody.

17. 17. The combination product of claim 15 or 16, wherein the unit dose of the second product is 0.1 to 100 mg, 0.5 to 50 mg, 1 to 20 mg, 2 to 15 mg, 4 to 12 mg, or 8 to 12 mg based on the mass of the active ingredients.

18. Use of a bispecific antibody according to any of claims 1 to 3, a conjugate according to claim 7, a pharmaceutical composition according to any of claims 10 to 14 or a combination product according to any of claims 15 to 17 in the preparation of a medicament for treating and / or preventing a tumor or anemia or for diagnosing a tumor or anemia.

19. A pharmaceutical agent for blocking the binding of PD-1 to PD-L1; A pharmaceutical agent for down-regulating the activity or level of PD-1; A pharmaceutical agent for alleviating immunosuppression of PD-1 in an organism; or A pharmaceutical agent for increasing the expression of IFN-γ and / or IL-2 in T lymphocytes; and / or A pharmaceutical agent for blocking the binding of CTLA4 to B7; A pharmaceutical agent for down-regulating the activity or level of CTLA4; A pharmaceutical agent for relieving immunosuppression of CTLA4 in an organism; or A pharmaceutical agent for increasing the expression of IL-2 in T lymphocytes; 18. Use of a bispecific antibody according to any of claims 1 to 3, a conjugate according to claim 7, a pharmaceutical composition according to any of claims 10 to 14, or a combination product according to any of claims 15 to 17 in the preparation of

20. A bispecific antibody according to any of claims 1 to 3, a conjugate according to claim 7, a pharmaceutical composition according to any of claims 10 to 14 or a combination product according to any of claims 15 to 17 for use in the treatment and / or prevention of tumors or anemia or for use in the diagnosis of tumors or anemia.

21. The bispecific antibody according to any of claims 1 to 3, the conjugate according to claim 7, the pharmaceutical composition according to any of claims 10 to 14 or the combination product according to any of claims 15 to 17, for use according to claim 20, wherein the bispecific antibody, the conjugate, the pharmaceutical composition or the combination product is for administration before or after a surgical treatment and / or before or after radiotherapy.

22. the unit dose of the bispecific antibody is 0.1 to 100 mg per kg of body weight; 22. The pharmaceutical composition of claim 21.

23. The bispecific antibody according to any of claims 1 to 3, the conjugate according to claim 7, the pharmaceutical composition according to any of claims 10 to 14 or the combination product according to any of claims 15 to 17, for use according to claim 20, wherein the bispecific antibody, the conjugate, the pharmaceutical composition or the combination product is for administration performed in 2 or 3 week cycles.

Images