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

Abstract

To provide anti-CTLA4 / anti-PD-1 bispecific antibodies and uses thereof in the field of tumor therapy and molecular immunology.SOLUTION: The anti-CTLA4 / anti-PD-1 bifunctional antibody comprises a first protein functional region targeting PD-1 and a second protein functional region targeting CTLA4 where the heavy chain constant region of the immunoglobulin comprised 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 to FcγRIIIa and C1q is reduced after the mutation as compared with the affinity constant before mutation. The bifunctional antibody well and specifically binds to CTLA4 and PD-1, capable of alleviating immunosuppression of CTLA4 and PD-1 in an organism and activating T lymphocytes with good application prospect.SELECTED DRAWING: Figure 13

Description

Technical Field

[0001] The present invention relates to the fields of tumor treatment and molecular immunology, and in particular, to anti-CTLA4 / anti-PD-1 bispecific antibodies and their use. More particularly, the present invention relates to mutant anti-CTLA4 / anti-PD-1 bispecific antibodies.

Background Art

[0002] The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed in activated T cells, B cells, and myeloid cells. Both ligands of PD-1, 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 in various cells including T cells, B cells, endothelial cells, and epithelial cells, and PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0003] The PD-1 / PDL1 signaling pathway plays an important role in the regulation of immune tolerance, microbial infection, and tumor immune escape. PD-1 is mainly expressed in immune cells, such as T cells, and the ligand of PD-1, PDL1, is highly expressed in multiple human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway activates inhibited T cells so that the T cells can attack cancer cells. Blocking PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, activate the tumor cell killing process, 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 effectiveness of PD-1 antibodies, antibodies targeting the PD-1 pathway are expected to bring breakthroughs in the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet M. B., 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), which is widely recognized in the industry.

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

[0005] A CTLA4 antibody (or anti-CTLA4 monoclonal antibody) or CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing the reactivity of T cells 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 treating 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 subsets and is an important factor in the regulation of 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 US FDA for treating malignant tumors, including melanoma, kidney cancer, etc., and clinical studies for treating chronic viral infections are currently underway (Chavez, A.R., et al., 2009, Ann. N.Y. Acad. Sci., 1182:p.14-27). CTLA4 and CTLA4 antibodies are important influencing factors for T cell function and interfere with the immune microenvironment in the body. CTLA4 antibodies can specifically relieve the immunosuppression of CTLA4, activate T cells, and induce IL-2 production, and in vitro and in vivo studies have demonstrated their promise in broad applications in gene therapy for diseases such as tumors and parasitic infections.

[0007] CTLA4 antibodies can produce specific therapeutic effects and significant effectiveness against diseases and can be used to supplement traditional drugs and explore new means of gene therapy.

[0008] Bispecific antibodies are also known as bifunctional antibodies, which 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 offers flexibility in terms of binding site optimization, synthetic form, yield, and allogeneic aspects, thus having certain advantages. Currently, more than 45 forms have been demonstrated (Dafne Muller, Kontermann R E., 2010, BioDrugs, 24(2):89-98). A number of developed bispecific antibodies are in the IgG-scFv form, 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 forms for bispecific antibodies due to its similarity to the naturally occurring IgG form and the advantages in antibody manipulation, 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 an antibody to an epitope of a virus-infected cell or tumor cell and the binding of the Fc fragment of the antibody to the Fc receptor (FcR) on the surface of the killer cell.

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

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

[0012] According to the types of antibodies recognized by Fc receptors and the types of expressing cells, Fc receptors are mainly classified into three types, FcγR, FcαR, and FcεR. 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 the ADCC effect. FcγRIII is the most dominant molecule mediating ADCC and has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. In the FcγRIIIa population, there are two subtypes, FcγRIIIa_V158 with high affinity and FcγRIIIa_F158 with low affinity, distinguished by single nucleotide polymorphism (SNP) sites. FcγRI has a higher affinity for the Fc region of IgG and participates in the ADCC process; FcγRII includes three subtypes, FcγRIIa, FcγRIIb, and FcγRIIc (also known as CD32a, CD32b, and CD32c, respectively), among which FcγRIIa has ADCC activity; for FcγRIIa, there are two subtypes, FcγRIIa_H131 and FcγRIIa_R131, present in humans due to single nucleotide mutations; FcγRIIb is an inhibitory receptor and a typical inhibitory FcγR that inhibits the nearby ITAM pathway. For example, after the binding of immune complexes to BCR, the Fc fragment binds to FcγRIIb on the same cell, negatively regulating B cell activation and reducing the secretion of antibodies and cytokines (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 the amino acids in the crystallizable fragment (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 can induce ADCC and CDC effects. IgG2 has the lowest affinity for FcγR but can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 is characterized by the highest binding ability to FcγR and can induce higher CDC effects than ADCC and IgG1. IgG4 molecules demonstrate weak binding to FcγRs other than FcγRI and have a lower probability of causing 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 immune cells and lead to pharmacological adverse effects. Currently, there is still 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 activities against immune cells to which anti-CTLA4 / anti-PD-1 bispecific antibodies bind, and to improve the effectiveness of antibody therapies.

[0014] Chemotherapy is currently mainly classified into the following nine classes (He Jie, et al., Clinical Oncology, Beijing, People’s Medical Publishing House, 2016:230-237). The first class is drugs that directly bind to DNA and prevent DNA replication, including various alkylating agents, mitomycin, bleomycin, dacarbazine, platinum-based drugs (such as cisplatin and carboplatin), camptothecin, and their derivatives. The second class is drugs for preventing nucleic acid biosynthesis that mainly affect the enzyme system 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 mainly act on cells in the S phase and are antimetabolic chemotherapeutic agents and cell cycle-specific anticancer drugs. The third class is chemotherapeutic agents that affect transcription through a pharmacological mechanism in which the drug is inserted into the DNA double helix to form a non-covalent bond with the DNA double helix, interfering with the transcription of genetic information on DNA into DNA-dependent mRNA, causing damaged template function and disrupted transcription. The fourth class is those that affect tubulin and mitosis, including vinca alkaloids, podophyllotoxin, and taxanes. The fifth class is drugs that affect the function of ribosomes to block protein synthesis; a representative of such drugs is harringtonine, which inhibits the initiation of protein synthesis, decomposes ribosomes, and releases new peptide chains, but does not block the binding of mRNA and tRNA to ribosomes; such drugs cause reduction of nuclear DNA and cytoplasmic RNA and depolymerization of polysomes, inhibiting mitosis. The sixth class is drugs that affect the membrane of tumor cells, such as concanavalin (Con-A) and phytohemagglutinin (PHA); they bind to glycoprotein receptors on the cell membrane, thereby being able to affect DNA synthesis in tumor cells and prevent tumor cells from dividing. The seventh class is drugs that induce apoptosis, such as arsenic trioxide.The eighth class are hormones that treat tumors by regulating the endocrine system, and this includes estrogen, anti-estrogen, progesterone, androgen, anti-androgen, corticosteroid, and anti-corticosteroid (including dichlorodiphenyldichloroethane and aminoglutethimide). The ninth class is anti-cancer targeted therapy, and this includes monoclonal antibodies, epidermal growth factor signal transduction inhibitors (e.g., targeted drugs against the receptor tyrosine kinase pathway), ubiquitin-proteasome inhibitors, and angiogenesis inhibitors.

[0015] Anlotinib is a quinoline derivative tyrosine kinase inhibitor. As a multi-target tyrosine kinase inhibitor (TKI), it affects tumor angiogenesis and proliferation signal transduction. The main targets include receptor tyrosine kinase vascular endothelial growth factor receptor (VEGFR) 1-3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR) 1-4, platelet-derived growth factor receptor (PDGFR) α and β, and stem cell factor receptor (SCFR) 7, 8, and 9. The phase 2 trial showed that anlotinib improved the progression-free survival period with potential benefits for overall survival (Han B, et al., Br J Cancer, 2018; 118(5):654-661). The multi-center double-blind randomized phase 3 clinical trial showed that anlotinib resulted in extended and progression-free survival in Chinese patients. The findings suggested that anlotinib has good tolerance and is a potential third-line or further treatment for patients with advanced NSCLC (Han B, et al., JAMA Oncol., 2018 Nov.; 4(11):1569-1575).

[0016] Example 24 of the pamphlet of International Publication No. 2008 / 112407 of Patent Document 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.

Chemical formula

[0017] The structure of lenvatinib, 4-(3-chloro-4(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, is disclosed in Example 368 of US Patent No. 7,612,208. US Patent No. 7,253,286 discloses the mesylate form of lenvatinib, named 4-[3-chloro-4-(cyclopropylureido)phenoxy]-7-methoxyquinoline-6-carboxamide mesylate (i.e., lenvatinib mesylate), and its chemical structure is provided below (Formula II):

Chemical formula

Summary of the Invention

[0018] Through intensive research and creative efforts, the inventor has correspondingly modified the Fc fragment of the anti-CTLA4 / anti-PD-1 antibody structure to reduce the binding ability of the Fc region to the Fc receptor, 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 present invention is described in detail below.

[0020] One aspect of the present invention is a bispecific antibody, 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 the immunoglobulin, the heavy-chain variable region contains HCDR1-HCDR3 of the amino acid sequences respectively set forth in SEQ ID NOs: 27-29, and the light-chain variable region contains LCDR1-LCDR3 of the amino acid sequences respectively set forth in SEQ ID NOs: 30-32; for the single-chain antibody, the heavy-chain variable region contains HCDR1-HCDR3 of the amino acid sequences respectively set forth in SEQ ID NOs: 33-35, and the light-chain variable region contains LCDR1-LCDR3 of the amino acid sequences respectively set forth in SEQ ID NOs: 36-38; Or, For an immunoglobulin, the heavy chain variable region contains HCDR1 - HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 33 - 35 respectively, and the light chain variable region contains LCDR1 - LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 36 - 38 respectively; for a single - chain antibody, the heavy chain variable region contains HCDR1 - HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 27 - 29 respectively, and the light chain variable region contains LCDR1 - LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 30 - 32 respectively; The immunoglobulin is an immunoglobulin of human IgG1 subtype; According to the EU numbering system, the heavy - chain constant region of the immunoglobulin has mutations at any two or three of positions 234, 235 and 237, and the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced after the mutation compared to that before the mutation; preferably, the affinity constant is measured by the Fortebio Octet system. Relates to a bispecific antibody.

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

[0022] In one or more embodiments of the present invention, for the bispecific antibody, according to the EU numbering system, the heavy - chain constant region of the immunoglobulin has the following mutations at position 234, 235 and / or 237: L234A and L235A; L234A and G237A; L235A and G237A; Or L234A, L235A and G237A Has.

[0023] In the present invention, unless otherwise specified, the character before the position number represents the amino acid before mutation, and the character 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 the immunoglobulin, 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 the single-chain antibody, 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 the immunoglobulin, 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 the single-chain antibody, 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 an immunoglobulin of human IgG1 subtype; According to the EU numbering system, the heavy-chain constant region of the immunoglobulin has the following mutations at positions 234, 235 and / or 237: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A having bispecific antibodies.

[0025] In one or more embodiments of the present invention, for bispecific antibodies, according to the EU numbering system, the heavy chain constant region of the immunoglobulin 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 having one or more mutations selected from

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

[0027] In one or more embodiments of the present invention, for bispecific antibodies 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; 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 is any one of the following (1) to (20): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 4; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 8; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 12; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is as set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 4; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 8; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 12; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 16; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 20; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 16; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 20; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 16; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 20; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 42; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 44; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 42; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 44; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 16; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 16; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 20; and, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is described in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is described in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 20 is selected from any one of them.

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

[0030] In one or more embodiments of the present invention, for the bispecific antibody, the immunoglobulin or its antigen-binding fragment is about 10 -7 M or higher, for example, 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 higher than or equal to M; preferably, the affinity constant is measured by the Fortebio Octet system; preferably, the immunoglobulin or its antigen-binding fragment has no binding signal to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb, or has a binding signal lower than 0.1 nm; preferably, the binding signal refers to the response measured by the Fortebio Octet system.

[0031] In one or more embodiments of the present invention, for the bispecific antibody, the immunoglobulin or its antigen-binding fragment is about 10 -9 higher than M, for example, about 10 -8 M, 10 -7 M, 10 -6 M, or higher than 10 -5 binds to C1q with an affinity constant higher than or equal to M; preferably, the affinity constant is measured by the Fortebio Octet system; preferably, the immunoglobulin or its antigen-binding fragment has no binding signal to C1q, or has a binding signal lower than 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 present invention, for the bispecific antibody, the first protein functional region is linked to the second protein functional region directly or via a linker fragment; and / or the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody directly or via a linker fragment.

[0033] In one or more embodiments of the present invention, for the bispecific antibody, 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 present invention, for the bispecific antibody, the number of the first protein functional region and the number of the second protein functional region are each independently one, two or more.

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

[0036] In one or more embodiments of the present invention, for the bispecific antibody, the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin. Since the 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 present invention, the constant region of the immunoglobulin is 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 present invention, the bispecific antibody has a K -5 lower than about 10 -6 M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower and binds to the CTLA4 protein and / or the PD-1 protein. D

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

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

[0041] In one or more embodiments of the present invention, the bispecific antibody comprises a first protein functional region that targets PD-1, and a second protein functional region that targets CTLA4 ; the number of the first protein functional regions is one, and the number of the 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 heavy chain of the immunoglobulin is described in SEQ ID NO: 40, and the amino acid sequence of the light chain of the immunoglobulin is described in SEQ ID NO: 24; the amino acid sequence of the heavy chain variable region of the single-chain antibody is described in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is described in SEQ ID NO: 44; the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; 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 described in SEQ ID NO: 26.

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

[0045] Another aspect of the present invention is a conjugate comprising an antibody or an antigen-binding fragment thereof, and a conjugated moiety, wherein the immunoglobulin is a bispecific antibody according to any embodiment of the present 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, and relates to the conjugate.

[0046] Another aspect of the present invention is a kit comprising a bispecific antibody according to any embodiment of the present invention or a conjugate of the present 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, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme, and relates to the kit.

[0047] Another aspect of the present invention relates to the use of a bispecific antibody or conjugate according to any embodiment of the present 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 present invention is a pharmaceutical composition comprising a bispecific antibody or conjugate according to any embodiment of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, and relates to the pharmaceutical composition.

[0049] In one or more embodiments of the present invention, the pharmaceutical composition further 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., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate).

[0050] In one or more embodiments of the present invention, the unit dose of the pharmaceutical composition 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.

[0051] Another aspect of the present invention is a combination product comprising a first product and a second product in separate packages, wherein the first product comprises a bispecific antibody, conjugate or pharmaceutical composition according to any embodiment of the present 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., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate); preferably, the first product and the second product each further comprise one or more pharmaceutically acceptable excipients; preferably, the combination product further comprises a package insert, relating to the combination product.

[0052] In one or more embodiments of the present invention, for the combination product, 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 present invention, for the combination product, the unit dose of the second product is 0.1-100 mg, 0.5-50 mg, 0.5-10 mg, 1-10 mg, 2-8 mg, or 1-5 mg based on the mass of the active ingredient.

[0054] In one or more embodiments of the present invention, for a combination product, 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 present invention is the use of a bispecific antibody, conjugate, pharmaceutical composition or combination product according to any embodiment of the present invention in the preparation of a medicament for treating and / or preventing a tumor or anemia, or in the preparation of a medicament for diagnosing a tumor or anemia; 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 cancer; preferably, the tumor is a solid tumor with an MSI-H / dMMR phenotype; preferably, the tumor is a colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma or ovarian germ cell neoplasm with an 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 the use.

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

[0057] MSI-H and dMMR represent the results of two different assays, are biologically consistent, and are referred to as MSI-H / dMMR or high-frequency MSI / dMMR, while MSI-L and MSS are phenotypes of proficient MMR (pMMR). Detection of dMMR is performed by immunohistochemistry of protein expression for four mismatch genes, MSH2, MLH1, MSH6, and PMS2, based on tumor specimens (including surgical specimens and aspirate specimens). Absence of any of the four proteins supports dMMR; positive results for all four proteins indicate pMMR, i.e., a complete mismatch repair function. Detection of MSI is to match the lengths of repetitive DNA sequences (microsatellite sequences) in tumor cells and somatic cells and to compare the lengths. 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 referred to as next-generation sequencing, or NGS) can also be used as a method for detecting microsatellite instability. When more microsatellite loci, e.g., more than five loci or other microsatellite loci, are selected, for the PCR assay, inconsistency at ≥ 30% of the loci is defined as MSI-H, consistency at all loci is defined as MSS, and inconsistency of 0 - 30% is defined as MSI-L.

[0058] Another aspect of the present invention is a medicament for blocking the binding of PD-1 to PD-L1, a medicament for downregulating the activity or level of PD-1, a medicament for alleviating the immunosuppression of PD-1 in an organism, or a medicament for increasing the expression of IFN-γ and / or IL-2 in T lymphocytes; and / or a medicament for blocking the binding of CTLA4 to B7, A medicament for downregulating the activity or level of CTLA4, a medicament for alleviating the immunosuppression of CTLA4 in an organism, or a medicament for increasing the expression of IL-2 in T lymphocytes relates to the use of a bispecific antibody or conjugate according to any embodiment of the present invention in the preparation thereof.

[0059] Another aspect of the present invention is a method for preventing and / or treating tumors and / or adjuvant treatment and / or diagnosis, 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 present 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 cancer; 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 with MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors with MSI-H / dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm selected from one or more of them, relates to the method.

[0060] In one or more embodiments of the present invention, for the method, the administration is carried out before or after surgical treatment and / or before or after radiotherapy.

[0061] In one or more embodiments of the present invention, in the method, The unit dose of the anti-CTLA4 / anti-PD-1 bispecific antibody is 0.1 to 100 mg, 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) per kg of body weight; 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) per subject, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg; Preferably, the dose is administered 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 intravenous injection.

[0062] In some embodiments, the administration of the anti-CTLA4 / anti-PD-1 bispecific antibody is carried out in a cycle of 2 weeks (14 days) or 3 weeks (21 days), 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] A bispecific antibody, conjugate, pharmaceutical composition, or combination product according to any embodiment of the invention for use in the prevention and / or treatment of tumors and / or adjuvant treatment and / or diagnosis; 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 cancer; 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 with an MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors with an MSI-H / dMMR phenotype: colorectal cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell neoplasm selected from one or more of Also provided are bispecific antibodies, conjugates, pharmaceutical compositions or combination products according to any embodiment of the invention for said use.

[0064] Blocking the binding of PD-1 to PD-L1, Downregulating the activity or level of PD-1, Relieving the immunosuppression of PD-1 in an organism, or Upregulating the expression of IFN-γ in T lymphocytes; and / or Blocking the binding of CTLA4 to B7, Downregulating the activity or level of CTLA4, Relieving the immunosuppression of CTLA4 in an organism, or Upregulating the expression of IL-2 in T lymphocytes Also provided are bispecific antibodies or conjugates according to any embodiment of the invention for use in

[0065] Antibody drugs, especially monoclonal antibodies (mAbs), have achieved good efficacy in the treatment of various diseases. Traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens and obtain antibodies that target the antigens in the immunized animals, or to improve those antibodies with lower affinity for the antigens 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, namely, complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, LCDR3; these are 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 the following (1) to (13) are analyzed by technical means well known to those skilled in the art, for example, by 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 described in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is described 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 described in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is described 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 described in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is described 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 those of BiAb001 (M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of 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 those of BiAb001 (M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of 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 those of BiAb001 (M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of BiAb001 (M).

[0077] For the antibody BiAb004(hG1TM) of the present invention, amino acid mutations are introduced into 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) is obtained by introducing a point mutation from leucine to alanine (L234A) at position 234, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237 in the hinge region of the heavy chain.

[0079] Unless otherwise defined herein, 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. Definitions and explanations of related terms are provided below to better understand the present invention.

[0080] As used herein, when referring to the amino acid sequence of the CTLA4 protein (cytotoxic T lymphocyte antigen 4), it includes the full length of the CTLA4 protein, or the extracellular fragment CTLA4ECD of CTLA4 or a fragment containing CTLA4ECD; fusion proteins of CTLA4ECD, such as fragments fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG, are also included. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally generated or artificially introduced 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 their natural or artificial variants. Additionally, when describing a sequence fragment of the CTLA4 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

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

[0082] As used herein, unless otherwise specified, B7 is B7-1 and / or B7-2; its specific sequence is known in the prior art and can be referred to the sequences disclosed in existing literature or GenBank. For example, B7-1 (CD80, NCBI Gene ID: 941); B7-2 (CD86, NCBI Gene ID: 942).

[0083] As used herein, EC 50 refers to the concentration for 50% of the maximum effect, i.e., the concentration that can cause 50% of the maximum 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, the heavy chain can be interpreted as the polypeptide chain having a larger molecular weight in the antibody, and the light chain refers to the polypeptide chain having a smaller molecular weight in the antibody. The light chains are classified as κ and λ light chains. The heavy chains are generally classified as μ, δ, γ, α, or ε, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region and the constant region are linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a 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 a 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. V H and V L regions can be further divided into highly variable regions (called complementarity-determining regions (CDRs)) with conserved regions called framework regions (FRs) distributed therebetween. Each V H and V L consists of three CDRs and four FRs organized in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L(a) Each forms an antibody-binding site. The amino acid assignment to each region or domain follows the definitions of 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, for example 6, 9, or 12 CDRs. For example, in the bispecific antibodies of the present invention, the heavy chain may be a 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 9 CDRs. The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of a different isotype, such as IgG (e.g., subtype 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 by 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 otherwise clearly defined in the context, when referring to the term "antibody", 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 that is derived from a group of highly homologous antibodies, i.e., a group of identical antibody molecules, except for naturally occurring mutations that may occur. Monoclonal antibodies are highly specific for a single epitope on an antigen. In contrast, polyclonal antibodies generally contain at least two or more different antibodies that generally identify different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using DNA recombination (see, e.g., U.S. Patent 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 by the CDR regions of a non-human antibody (donor antibody), and the donor antibody 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 by the amino acid residues of the corresponding non-human antibody or by the amino acid residues of another antibody to further improve or optimize the performance of the antibody. 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. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants generally consist of chemically active surface groups of a molecule, such as amino acids, carbohydrates, or sugar side chains, and usually have unique three-dimensional structural features and unique charge features. For example, an epitope generally contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation that can generally be "linear" or "conformational". See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. 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 each other.

[0090] As used herein, the term "isolated" refers to being obtained by artificial means from its natural state. If a particular "isolated" substance or component is found in nature, it may be the case that a change occurs in its natural environment, or that it is isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not a particular isolated one exists naturally in a particular living animal, and the same polynucleotide or polypeptide having a high purity isolated from such a natural state is called 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 E. coli (strain) and a vector, and the E. coli (strain) is derived from commercially available strains, including but not limited to, for example, GI698, ER2566, BL21(DE3), B834(DE3), and BLR(DE3).

[0092] As used herein, the term "vector" refers to a nucleic acid medium into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element 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. Examples of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector 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, a vector may further contain an origin of replication.

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

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

[0095] As used herein, the term "K D " 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 stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, for example, when measured by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio Octet system, an antibody has a dissociation equilibrium constant (K -5 lower than about 10 -6 M, such as about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M or lower. 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; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also, in this specification, 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 carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington’s Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania, Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and the ionic strength enhancer includes, but is not limited to, sodium chloride.

[0098] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that can enhance the immune response of an organism to an antigen or change the type of immune response when delivered to the organism with or prior 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, lipopolysaccharides, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more used in clinical trials.

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

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

[0101] A "metastatic" cancer refers to a cancer that spreads from one part of the body (e.g., the lung) to another.

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

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

[0104] In the present invention, "about" or "approximately" means that the defined value or physical quantity varies within a range of 10%, 20% or 30% if not otherwise specified. For example, about 100 minutes or approximately 100 minutes can be 90 minutes to 110 minutes, 80 minutes to 120 minutes or 70 minutes to 130 minutes.

[0105] Advantages of the present invention: The present invention achieves one or more of the following technical effects (1) to (3): (1) The modification of the Fc fragment of the antibody of the present invention by the present inventor completely eliminates the binding activity of BiAb004 (hG1TM) to FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIIa_F158, thereby eliminating the ADCC activity and / or ADCP activity. (2) The modification of the Fc fragment of the antibody of the present invention by the present inventor completely eliminates the binding activity to complement C1q, thereby eliminating the CDC activity. (3) The antibody of the present invention has the potential ability for use in the preparation of a medicament for preventing and treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0106]

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Mode for Carrying Out the Invention

[0107] Detailed Description 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 used only to illustrate examples of the present invention and should not be considered as limiting the scope of the present invention. When technologies or conditions are not specified, the examples are carried out according to the technologies or conditions described in the literature in the relevant technical field (for example, refer to 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 manuals. The reagents or equipment used are conventional products commercially available when the manufacturer is not specified.

[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 the informed consent of the donor. Raji-PDL1 is a cell that expresses human PD-L1 constructed by transfection based on human B cell Raji by Akeso Biopharma. 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 the Fab F10.6.6 sequence in the study reported by Acierno et al. under the title "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 (registered trademark) 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 (registered trademark), and it 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 the same as those of 4G10, 4G10H1L1, and 4G10H3L3 respectively in Chinese Patent Publication No. 106967172(A).

[0110] (1) Variable Region Sequences of the Heavy and Light Chains of 4G10 Nucleotide sequence of the heavy chain variable region: (372bp) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAGTTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO: 1) Encoded amino acid sequence: (124aa) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2) Nucleotide sequence of the light chain variable region: (378bp) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (SEQ ID NO: 3) Encoded amino acid sequence: (126aa) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4)

[0111] (2) Nucleotide sequences of the heavy and light chain variable regions of the humanized monoclonal antibody 4G10H1L1 Nucleotide sequence of the heavy chain variable region (4G10H1V): (345bp) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACCAACTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT(SEQ ID NO: 5) Encoded amino acid sequence: (115aa) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO: 6) Nucleotide sequence of the light chain variable region (4G10L1V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAATCGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG(SEQ ID NO: 7) Encoded amino acid sequence: (109aa) 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): (345 bp) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAACTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (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): (327 bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (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 the same as those of 14C12 and 14C12H1L1 respectively in Chinese Patent Publication Gazette No. CN106967172(A).

[0114] (1) Variable region sequences of the heavy and light chains of 14C12 Nucleotide sequence of the heavy chain variable region: (354bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC(SEQ ID NO: 13) Encoded amino acid sequence: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA(SEQ ID NO: 14) Nucleotide sequence of the light chain variable region: (321bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG(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) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 17) Encoded amino acid sequence: (118 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18) Nucleotide sequence of the light chain variable region: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 19) Encoded amino acid sequence: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 20) DNA sequence of 14C12H1 heavy chain (14C12H1): (1344bp) Encoded amino acid sequence: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) DNA sequence of the 14C12H1 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 patterns of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) are in the Morrison format (IgG-scFv), that is, the C-termini of the two heavy chains of one IgG antibody are separately linked via a linker fragment to the scFv fragment of another antibody. The components for the design of the heavy and light chains are shown in Table A below. [Table 1] In the above Table A: 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 the above Table A, 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 the 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 effectiveness.

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

[0119] For the purpose of distinguishing from the mutated antibody, hereinafter, BiAb004(M) is also referred to as BiAb004(hG1WT) in this example. The above BiAb004(M) is "wild-type" and contains the Ig gamma-1 chain C region (Accession: P01857) as the heavy chain constant region and the Ig kappa chain C region (Accession: P01834) as the light chain constant region.

[0120] Preparation Example 4: Design of amino acid mutations in non-variable regions based on the humanized bispecific antibody BiAb004 Based on BiAb004(hG1WT) obtained in Preparation Example 3, point mutations of leucine to alanine (L234A), leucine to alanine (L235A) at position 235, and glycine to alanine (G237A) at position 237 were introduced into the heavy chain to obtain BiAb004(hG1TM).

[0121] DNA sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1TM): (1344bp, positions of mutations are underlined) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGAGCC 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) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA 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 of the light chain. The specific sequences are shown in Preparation Example 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 therapeutic monoclonal antibodies to Fc receptors affects the safety and efficacy of the antibodies.

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

[0126] The method for determining the affinity constant of an antibody for FcγRI by the Fortebio Octet system can be briefly described as follows: The sample dilution buffer was a solution of 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. Association and dissociation constants of the antibody for FcγRI in buffer were determined using antibody concentrations of 3.12 - 50 nM (serial two-fold dilutions). The sensor with immobilized antigen was equilibrated in buffer for 60 s, then binding of the immobilized FcγRI on the sensor to the antibody was determined for 120 s; dissociation of FcγRI from the antibody was determined at 120 s. The temperature was 30 °C and the frequency was 0.3 Hz. The data was fitted and analyzed using a 1:1 model to obtain the affinity constant of the antibody for FcγRI.

[0127] Results of the affinity constant assays of BiAb004 (hG1TM) and BiAb004 (hG1WT) for FcγRI are shown in Table 1 and Figures 1 and 2 below.

Table 2

[0128] The results suggest that the binding activity of BiAb004 (hG1TM) for FcγRI was 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) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect.

[0130] In this experiment, the affinity constants of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIIa_V158 were measured 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 by the Fortebio Octet system can be briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and the binding of the immobilized FcγRIIIa_V158 on the sensor to the antibody at concentrations of 31.25 - 500 nM (serial two-fold dilution) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was regenerated 4 times for 5 s each in 10 mM glycine (pH 1.5). 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 assays of BiAb004 (hG1TM) and BiAb004 (hG1WT) for FcγRIIIa_V158 are shown in Table 2 below and Figures 3 and 4.

Table 3

[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) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect.

[0135] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 were measured 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 by 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. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and the binding of the immobilized FcγRIIIa_F158 on the sensor to the antibody was determined for 60 s at concentrations of 31.25 - 500 nM (serial two-fold dilution). The antibody was dissociated in the buffer for 60 s. The sensor was regenerated 4 times for 5 s each in 10 mM glycine (pH 1.5). The temperature was 30 °C and the frequency was 0.3 Hz. The data was analyzed by fitting to a 1:1 model to obtain the affinity constant.

[0137] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIIa_F158 are shown in Table 3 and Figures 5 and 6 below.

Table 4

[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) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect.

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

[0141] The method for determining the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_H131 by the Fortebio Octet system can be briefly described as follows: The immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, 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 a 300 s blocking in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, and the binding of the immobilized FcγRIIa_H131 on the sensor to the antibody was determined for 60 s at concentrations of 12.5 - 200 nM (serial two-fold dilutions). The antibody was dissociated for 60 s in the buffer. 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 was analyzed by fitting to a 1:1 model to obtain the affinity constants.

[0142] The results of the affinity constant assays of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIa_H131 are shown in Table 4 below and Figures 7 and 8.

Table 5

[0143] The results suggest that the binding activity of BiAb004(hG1TM) for FcγRIIa_H131 was 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) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect.

[0145] In this experiment, the affinity constants of BiAb004 (hG1WT) and BiAb004 (hG1TM) for FcγRIIa_R131 were measured 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) by the Fortebio Octet system can be briefly described as follows: The immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, 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 a 300 s blocking in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, and the binding of the immobilized FcγRIIa_R131 on the sensor to the antibody was determined for 60 s at concentrations of 12.5 - 200 nM (serial two-fold dilutions). The antibody was dissociated 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 was analyzed by fitting to a 1:1 model to obtain the affinity constants.

[0147] The results of the affinity constant assay of BiAb004 (hG1TM) and BiAb004 (hG1WT) for FcγRIIa_R131 are shown in Table 5 and Figures 9 and 10 below.

Table 6

[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 showed that BiAb004(hG1WT) has a stronger binding signal and thus 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) can bind to the Fc fragment of IgG antibodies, downregulate the functions of immune cells, inhibit the activation and proliferation of immune cells, and inhibit the secretion of cytokines.

[0150] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb were measured 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 by the Fortebio Octet system is briefly described as follows: The immobilization dilution buffer is a solution of PBS, 0.02% Tween-20, 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 a 300 s blocking in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4, and the binding of the immobilized FcγRIIb on the sensor to the antibody was determined for 60 s at concentrations of 12.5 - 200 nM (serial two-fold dilutions). The antibody was dissociated 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 was analyzed by fitting to a 1:1 model to obtain the affinity constant.

[0152] The results of the affinity constant assays of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIb are shown in Table 6 and Figures 11 and 12 below.

Table 7

[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. The binding of therapeutic monoclonal antibodies to C1q affects the safety and efficacy of the antibodies.

[0154] In this experiment, the affinity constants of BiAb004 (hG1WT) and BiAb004 (hG1TM) for C1q were measured using the Fortebio Octet system to evaluate the CDC activity of the antibodies. In this experimental example, the anti-PDL1 antibody 5C10H2L2-IgG1mt was used as a control, and its preparation is described in the pamphlet of PCT International Publication No. 2017148424 (A1).

[0155] The method for determining the affinity constant of an antibody for C1q by 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. Antibodies at 50 μg / mL were immobilized on the FAB2G sensor at a fixed height of approximately 2.0 nm. The sensor was equilibrated for 60 s in the buffer for blocking, and the binding of the immobilized antibody on the sensor to the antigen C1q at concentrations of 1.25 - 20 nM (serial two-fold dilution) was determined for 60 s. The antigen and antibody were dissociated in the buffer for 60 s. The sensor was regenerated 4 times for 5 s each in 10 mM glycine (pH 1.7). The shaking speed of the sample plate was 1000 rpm, 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 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 assays of BiAb004 (hG1TM), BiAb004 (hG1WT), and 5C10H2L2-IgG1mt for C1q are shown in Table 7 below and Figures 13, 14, and 15.

Table 8

[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 effector immune cells with killing activity recognizing the Fc fragment of an antibody bound to an antigen of a target cell through Fc receptors (FcR) expressed on their surface and directly killing the target cell. To test the ADCC effect of the anti-CTLA4 / anti-PD-1 bifunctional antibodies BiAb004(hG1WT) and BiAb004(hG1TM), the inventors constructed a co-culture system of 293T-CTLA4-PD1 cells and primary PBMCs expressing PD-1 and CTLA4 antigens to test the ADCC activity of the antibodies.

[0159] The method for the ADCC activity assay of 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 instructions for using Ficoll to isolate peripheral blood mononuclear cells. 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 was 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; hereinafter referred to as medium), and washed twice. The cells were counted and viability was determined. The cell density was adjusted to an appropriate range using the medium. According to the experimental design, 100 μL of 293T-CTLA4-PD1 cells were added to a 96-well plate at a density of 3.0E+04 cells / well. 50 μL of antibody was added and the cells were pre-incubated 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 incubated in a carbon dioxide incubator at 37 °C with 5% CO2 for 4 h. The cells were centrifuged at 250×g for 5 minutes. 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 was added to each well according to the instructions for using the Cytotoxicity Detection Kit. The cells were incubated at room temperature in the dark for 30 minutes. The OD values at 490 nm and 650 nm were measured. The ADCC percentage for each group was calculated according to the formula ADCC (%) = (treatment group - negative control group) / (maximum LDH release in target cells - spontaneous LDH release in target cells) × 100%.

[0160] The ADCC activities of BiAb004 (hG1WT) and BiAb004 (hG1TM) in 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens are represented as ADCC percentages, and the results are shown in Figure 16.

[0161] In the co-culture system of PBMC and 293T-CTLA4-PD1, the results showed that the percentage of ADCC induced by BiAb004 (hG1TM) was significantly lower than that induced by the same level of BiAb004 (hG1WT).

[0162] The results suggest that BiAb004 (hG1TM) has no ADCC activity.

[0163] Experimental Example 9: Inhibition of the growth of colorectal cancer MC38 cell xenografts in mice by BiAb004 (hG1TM) combined with lenvatinib The mouse MC38 cell line is a mouse 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 collected, adjusted to a density of 5 million cells / mL, and transplanted subcutaneously into the right flank of PD-1 transgenic mice (purchased from Shanghai Model Organisms Center, Inc.) at a volume of 200 μL / mouse. Transplantation was performed on 12 mice, each of which carried 1×10 6 cells. When the tumor volume reached approximately 60 - 150 mm 3 , the mice were divided into two groups, a model group and a BiAb004 (hG1TM) / lenvatinib treatment group, with 6 mice in each group based on the tumor volume (having a similar average volume of approximately 94 mm 3 ). The day of grouping was set as D0. Six doses of the antibody were administered intraperitoneally on D0, D4, D7, D10, D13, and D17 respectively; lenvatinib was administered orally by gavage once a day for 20 days. The tumor volume was measured with calipers. After grouping, the tumor dimensions were measured twice a week using calipers, and the formula TV = 0.5×ab 2The tumor 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 tumor volume). The specific protocol is shown in Table 8.

Table 9

[0165] The experimental results are shown in Figure 17.

[0166] BiAb004 (hG1TM) combined with lenvatinib significantly inhibited the growth of MC38 tumors; the results showed that BiAb004 (hG1TM) combined with lenvatinib was significantly effective against rectal and / or colon cancers with the MSI-H / dMMR phenotype.

[0167] Experimental Example 10: Pharmacodynamic evaluation of BiAb004 (hG1TM) in a C57BL / 6-hPD1 / hPDL1 / hCD73 mouse colon cancer MC38-hPDL1 / hCD73 subcutaneous xenograft tumor model Female C57BL / 6-hPD1 / hPDL1 / hCD73 mice (purchased from Nanjing GemPharmatech Co., Ltd.) were divided into 8 groups, and colon cancer MC38-hPDL1 / hCD73 cells (purchased from Nanjing GemPharmatech Co., Ltd.) (2×10 6(Cells / 100 μL / mouse) were transplanted. The day of transplantation was defined as D0. The dosing volume was adjusted according to body weight to 10 μL per 1 g of mouse body weight. An isotype control antibody (the preparation and supplier were the same as those in Experimental Example 9) or BiAb004 (hG1TM) was administered intraperitoneally twice a week for 3 weeks, for a total of 6 doses. Tumors were measured continuously during the experiment, and the following formula: Tumor volume (mm 3 ) = (Tumor length × (Tumor width) 2 ) / 2 was used to calculate the volume.

[0168] The results are shown in Figure 18 and Table 9 below.

Table 10

[0169] Experimental Example 11: Study on the antibody-mediated phagocytosis 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 the binding of the Fc fragment of antibodies targeting immune checkpoint molecules such as PD-1 and CTLA-4 to Fc receptors may potentially cause antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), which are important mechanisms leading to immune cell damage and may negatively affect antibody-mediated anti-cancer activity.

[0170] To test the ADCP activity of BiAb004(hG1TM), mouse macrophages were used as effector cells and cell lines overexpressing the corresponding antigen were used as target cells. The cell-mediated ADCP effect was tested. The femur marrow of C57BL / 6 mice (purchased from Guangdong Medical Laboratory Animal Center) was first aseptically collected and lysed on ice for 5 minutes with erythrocyte lysis buffer. Lysis was terminated using 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 an effective concentration of 100 ng / mL. The cells were cultured for 7 days at 37 °C and 5% CO2 in a cell culture chamber for induction. Half of the medium was replaced on days 3 and 5, and M-CSF was added. Induction of the cells was completed on day 7. The cells were digested using 0.25% trypsin. The macrophages were collected and centrifuged at 170×g for 5 minutes. The supernatant was discarded, the cells were suspended in DMEM complete medium, and counted. The cells were adjusted to an appropriate density and filled into sterile EP tubes for further use.

[0171] Logarithmic phase CHO-K1-CTLA4-PD1 cells (a cell line based on CHO-K1 cells overexpressing both human CTLA4 and PD1 antigens, constructed by Akeso Biopharma, Inc.) were collected, centrifuged at 170×g for 5 minutes, washed with PBS, resuspended, counted, and viability was determined. 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. 6 mL of complete DMEM medium was added to stop the staining. The cells were centrifuged at 170×g for 5 minutes and the supernatant was discarded. 1 mL of complete DMEM medium was added for resuspension. The cells were incubated in the incubator for 10 minutes and adjusted to the experimental density. The cells were coded as CHO-K1-PD1-CTLA4-CFSE. The test antibodies were diluted in complete DMEM medium. Isotype control antibody anti-HEL antibody and medium were used as the isotype control group and blank control group. According to the study design, the diluted antibodies 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 the working concentration of the antibody was 50, 5, and 0.5 nM). The mixture was resuspended, mixed uniformly, and incubated in the incubator at 37 °C for 2 h. 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 / 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 loading tubes and analyzed by a BD FACSCalibur flow cytometer. Macrophages in the system were APC+ Macrophages that were positive and involved in phagocytosis were double positive for APC and CFSE. The phagocytosis rate was determined as the ratio of the number of double positive cells to the number of APC positive cells, and the antibody-mediated ADCP activity was evaluated.

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

Number

[0173] In the antibody validation system for the mediation of phagocytosis activity, the results showed that the phagocytosis activity of CHO-K1-PD1-CTLA4 cells by macrophages mediated by BiAb004 (hG1TM) had no significant difference compared to that of the isotype control antibody anti-HEL antibody compared to BiAb004 (hG1WT), indicating that BiAb004 (hG1TM) has no ADCP activity.

[0174] The results suggest that the amino acid mutations introduced by BiAb004 (hG1TM) can effectively eliminate the ADCP effect, obtaining a surprising technical effect.

[0175] Experimental Example 12: Significantly enhanced immune response of immune cells against human gastric cancer KATO III cells by BiAb004 (hG1TM) Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Plus reagent, the isolated PBMCs were counted and frozen. Raji-PDL1 and KATO III cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM + 10% FBS complete medium. The PBMCs were thawed and activated with 0.5 μg / mL of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of mitomycin C (MMC) for 1 h. The 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing KATO III cells were collected and seeded on a 96-well plate at 5×10 4 cells / well. The diluted antibodies were added according to the research design. The mixture was mixed uniformly and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0176] The results are shown in Figure 20.

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

[0178] Experimental Example 13: Significantly enhanced immune response of immune cells against human cervical cancer Hela cells by BiAb004 (hG1TM) Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the instructions for use of Plus reagent, and the isolated PBMCs were counted and frozen. Raji-PDL1 and Hela cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. The PBMCs were thawed and activated with 0.5 μg / mL of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Hela cells in the logarithmic growth phase were collected and seeded on a 96-well plate at 5×10 4 cells / well. The diluted antibodies were added according to the research design. The mixture was uniformly mixed and incubated in a 5% CO2 incubator at 37 °C for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0179] The results are shown in Figure 21.

[0180] BiAb004 (hG1TM) more 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). Therefore, the results indicated that BiAb004 (hG1TM) is promising for the treatment of cervical cancer.

[0181] Experimental Example 14: Significantly enhanced immune response of immune cells against human T cell lymphoma Jurkat cells by BiAb004 (hG1TM) Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Plus reagent, the isolated PBMCs were counted and frozen. Raji-PDL1 and human T cell lymphoma Jurkat cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. The PBMCs were thawed and activated with 0.5 μg / mL of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of MMC for 1 h. The 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing Jurkat cells were collected and seeded on a 96-well plate at 1×10 5 cells / well. The diluted antibodies were added according to the study design. The mixture was uniformly mixed and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0182] The results are shown in Figure 22.

[0183] BiAb004 (hG1TM) significantly enhanced the immune response of immune cells against human T cell lymphoma Jurkat cells, as characterized by a significantly increased IL-2 secretion level compared to BiAb004 (hG1WT) while excluding ADCC and CDC (i.e., ADCP); the antibody had equivalent or higher pharmacological activity at doses of 1.34 nM and 20 nM compared to BiAb004 (hG1WT) and is promising for treating cervical cancer.

[0184] Experimental Example 15: Significantly enhanced immune response of immune cells against human nasopharyngeal carcinoma CNE-2Z cells by BiAb004 (hG1TM) Ficoll-PaqueTM PBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Plus reagent, 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 of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing CNE-2Z cells were collected and seeded on a 96-well plate at 3×10 4 cells / well. Diluted antibodies were added according to the research design. The mixture was uniformly mixed and incubated in a 5% CO2 incubator at 37 °C for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0185] The results are shown in Figure 23.

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

[0187] The above results showed that BiAb004(hG1TM) had better or equivalent pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC and ADCP effects, indicating the potential ability to treat human nasopharyngeal carcinoma.

[0188] Experimental Example 16: Significantly Enhanced Immune Response of Immune Cells against Human Breast Cancer MDA-MB-231 Cells by BiAb004(hG1TM) Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Plus reagent, 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. The PBMCs were thawed and activated with 0.5 μg / mL of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of MMC for 1 h. The 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing MDA-MB-231 cells were collected and seeded on a 96-well plate at 3×10 4 cells / well. The diluted antibody was added according to the research design. The mixture was uniformly mixed and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0189] The results are shown in Figure 24.

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

[0191] The above results indicated that BiAb004(hG1TM) has better or equivalent pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC, and ADCP effects, indicating its potential ability to treat human breast cancer.

[0192] Experimental Example 17: Significantly enhanced immune response of immune cells against human mesothelioma NCI-H2452 cells by BiAb004(hG1TM) Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the instructions of the Ficoll-Paque Plus reagent, counted, and frozen. Raji-PDL1 and NCI-H2452 cells (purchased from Shanghai Cell Bank, Chinese Academy of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing NCI-H2452 cells were collected and seeded on a 96-well plate at 3×10 4 cells / well. Diluted antibodies were added according to the research design. The mixture was uniformly mixed and incubated in a 5% CO2 incubator at 37°C for 3 days. After 3 days, cell culture supernatants were collected and tested for IL-2 according to the instructions of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0193] The results are shown in Figure 25.

[0194] BiAb004(hG1TM) significantly enhanced the immune response of immune cells against human mesothelioma cell line NCI-H2452 as characterized by significantly increased IL-2 secretion levels compared to BiAb004(hG1WT), thus the results indicated that BiAb004(hG1TM) 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(hG1TM) in combination with anlotinib Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the instructions of the Ficoll-Paque Plus reagent, 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 of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing A549 cells (purchased from Chinese Academy of Sciences Cell Bank) were collected and seeded on a 96-well plate at 5×10 4 cells / well. Diluted antibodies were added according to the study design. The mixture was uniformly mixed and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0196] As shown in Figure 26, BiAb004(hG1TM) 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 levels of IL-2 secretion compared to BiAb004(hG1TM) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) in combination with anlotinib hydrochloride. Therefore, BiAb004(hG1TM) 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(hG1TM) in Combination with Anlotinib Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Ficoll-Paque Plus reagent, the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H446 cells (purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. The PBMCs were thawed and activated with 0.5 μg / mL of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmic growth phase NCI-H446 cells (purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were collected and seeded on a 96-well plate at 8×10 4Cells were seeded in wells. Diluted antibodies and anlotinib were added according to the study design. The mixture was uniformly mixed and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0198] The results are shown in Figure 27.

[0199] BiAb004(hG1TM) 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 secretion levels of IL-2 compared to BiAb004(hG1MT) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) in combination with anlotinib hydrochloride. Therefore, BiAb004(hG1TM) 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(hG1TM) in combination with anlotinib Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the instruction manual of the Plus reagent, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H226 cells (purchased from 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 of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing NCI-H226 cells were collected and seeded on a 96-well plate at 5×10 4 cells / well. Diluted antibodies and anlotinib were added according to the study design. The mixture was evenly mixed and incubated in a 5% CO2 incubator at 37 °C for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instruction manual of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0201] The results are shown in Figure 28.

[0202] The BiAb004(hG1TM) monotherapy more effectively and significantly enhanced the immune response of immune cells against human squamous cell lung cancer NCI-H226 cells, as characterized by a significantly increased IL-2 secretion level compared to the BiAb004(hG1WT) monotherapy.

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

[0204] Based on the effective elimination of ADCC, CDC and ADCP effects, BiAb004(hG1TM) showed better or equivalent pharmacological activity compared to BiAb004(hG1WT) monotherapy or BiAb004(hG1WT) in combination with anlotinib, indicating better efficacy for treating human squamous cell lung cancer. The above results showed this.

[0205] Experimental Example 21: Significantly enhanced immune response of immune cells against MSI-H / dMMR phenotype human colorectal cancer SW48 cells by BiAb004(hG1TM) SW48 is a human colorectal cancer cell line and is identified with the MSI-H / dMMR phenotype (Branch P et al., (1995), Cancer Res, 55(11): 2304-2309.). It was used to detect the enhanced immune cell response against tumors with the MSI-H / dMMR phenotype by BiAb004(hG1TM).

[0206] In the experiment, Ficoll-Paque TMPBMCs were isolated from healthy human peripheral blood according to the instructions for use of Plus reagent, 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 of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing SW48 cells were collected and seeded on a 96-well plate at 2×10 5 cells / well. The diluted antibodies were added according to the study design. The mixture was uniformly mixed and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the instructions for use of the ELISA KIT. All media in this experiment were 10% FBS + RPMI 1640.

[0207] The results are shown in Figure 29.

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

[0209] BiAb004(hG1TM) has better or equivalent pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC, and ADCP effects, indicating the potential to treat solid tumors with MSI-H / dMMR phenotypes, particularly colorectal cancer and / or rectal cancer with MSI-H / dMMR phenotypes, as shown by the above results.

[0210] Experimental Example 22: Significantly enhanced immune response of immune cells against human colorectal cancer SW837 cells with MSI-H / dMMR phenotype by BiAb004(hG1TM) 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 it was used in this example to detect the enhanced immune cell response against tumors with non-MSI-H / dMMR phenotypes by BiAb004(hG1TM).

[0211] Ficoll-Paque TM PBMCs were isolated from healthy human peripheral blood according to the instructions for use of the Plus reagent, 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 of SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL of 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 seeded on a 96-well plate at 1×10 5 cells / well. Logarithmically growing SW837 cells were collected and seeded on a 96-well plate at 5×10 4Cells were seeded in wells. Diluted antibodies were added according to the study design. The mixture was uniformly mixed and incubated at 37 °C in a 5% CO2 incubator for 3 days. After 3 days, cell culture supernatants were collected and tested for IL-2 according to the instructions of the ELISA KIT.

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

[0213] The results are shown in Figure 30.

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

[0215] The above results indicated that BiAb004(hG1TM) had better or comparable pharmacological activity compared to BiAb004(hG1WT) based on the effective elimination of ADCC, CDC or ADCP effects, and pointed to its potential ability to treat solid tumors with non-MSI-H / dMMR phenotype, especially colorectal cancer 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 those details in accordance with all the disclosed teachings, and all these changes are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A bispecific antibody, 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; For the 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 the 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; the immunoglobulin is a human IgG1 subtype immunoglobulin; the immunoglobulin heavy chain constant region is mutated at any two or three of positions 234, 235 and 237 according to the EU numbering system, and the bispecific antibody has reduced antibody-dependent cellular phagocytosis (ADCP) and / or reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or reduced complement-dependent cytotoxicity (CDC) compared to an effective amount of a bispecific antibody that does not have the mutations at any two or three of positions 234, 235 and 237, Bispecific antibodies.

2. The bispecific antibody of claim 1 , wherein the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

3. 3. The bispecific antibody of claim 2, wherein 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.

4. 4. The bispecific antibody of claim 3, 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.

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

26.

6. The heavy chain constant region of the immunoglobulin is, according to the EU numbering system: 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 6. The bispecific antibody of claim 1 , further comprising one or more mutations selected from:

7. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 6.

8. A vector comprising the isolated nucleic acid molecule of claim 7.

9. A host cell comprising the isolated nucleic acid molecule of claim 7 or the vector of claim 8.

10. 10. A conjugate comprising a bispecific antibody and a conjugated moiety, wherein the bispecific antibody is the bispecific antibody of any one of claims 1 to 6, and the conjugated moiety is a detectable label, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

11. A kit comprising the bispecific antibody of any one of claims 1 to 6 or the conjugate of claim 10, The kit further comprises a second antibody capable of specifically recognizing the bispecific antibody or the conjugate.

12. The kit of claim 11 , wherein the second antibody further comprises a detectable label, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

13. 11. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 6 or the conjugate of claim 10, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

14. 14. The pharmaceutical composition of claim 13, further comprising one or more anti-tumor chemotherapeutic agents.

15. 15. The pharmaceutical composition of claim 14, wherein the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor.

16. the tyrosine kinase inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, or lenvatinib or a pharmaceutically acceptable salt thereof; 16. The pharmaceutical composition of claim 15.

17. 17. The pharmaceutical composition according to any one of claims 13 to 16, wherein 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.

18. A combination product comprising a first product and a second product in separate packages, the first product comprises a bispecific antibody according to any one of claims 1 to 6, a conjugate according to claim 10, or a pharmaceutical composition according to any one of claims 13 to 17; A combination product, wherein said second product comprises one or more anti-tumor chemotherapeutic agents.

19. 19. The combination product of claim 18, wherein the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor.

20. 20. The combination product of claim 19, wherein the tyrosine kinase inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, or lenvatinib or a pharmaceutically acceptable salt thereof. Combination products.

21. 21. The combination product of any one of claims 18 to 20, wherein the unit dose of the first product 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.

22. 22. The combination product of any one of claims 18 to 21, 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.

23. Blocking the binding of PD-1 to PD-L1; Downregulation of PD-1 activity or levels; Alleviation of PD-1 immunosuppression in the organism; or Increased expression of IFN-γ and / or IL-2 in T lymphocytes; and / or Blocking CTLA4 binding to B7; Downregulation of CTLA4 activity or levels; Alleviation of CTLA4 immunosuppression in the organism; or Increased expression of IL-2 in T lymphocytes; 23. A bispecific antibody according to any one of claims 1 to 6, a conjugate according to claim 10, a pharmaceutical composition according to any one of claims 13 to 17, or a combination product according to any one of claims 18 to 22, for use in

24. A bispecific antibody according to any one of claims 1 to 6, a conjugate according to claim 10, a pharmaceutical composition according to any one of claims 13 to 17 or a combination product according to any one of claims 18 to 22 for use in the treatment and / or prevention of tumors or anemia.

25. A bispecific antibody according to any one of claims 1 to 6, a conjugate according to claim 10, a pharmaceutical composition according to any one of claims 13 to 17, or a combination product according to any one of claims 18 to 22 for use in the diagnosis of tumors or anemia.

26. 26. The bispecific antibody, pharmaceutical composition or combination product for use according to claim 24 or 25, 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.

27. 27. The bispecific antibody, conjugate, pharmaceutical composition or combination product for use according to claim 26, wherein the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer.

28. 27. The bispecific antibody, conjugate, pharmaceutical composition or combination product for use according to claim 26, wherein the gastric cancer is adenocarcinoma or gastroesophageal junction adenocarcinoma.

29. 27. The bispecific antibody, conjugate, pharmaceutical composition or combination product for use according to claim 26, wherein said tumor is a solid tumor of MSI-H / dMMR phenotype.

30. The solid tumor of the MSI-H / dMMR phenotype is Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasms 30. The bispecific antibody, conjugate, pharmaceutical composition or combination product for use according to claim 29 selected from one or more of:

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