Anti-ctla4-anti-pd-1 bispecific antibodies, pharmaceutical compositions, and uses

Abstract

The application belongs to the field of tumor treatment and molecular immunology, and relates to an anti-CTLA4-anti-PD-1 bispecific antibody, a pharmaceutical composition and purposes. Specifically, the anti-CTLA4-anti-PD-1 bispecific antibody comprises a first protein functional region targeting PD-1 and a second protein functional region targeting CTLA4, wherein the heavy chain constant region of the immunoglobulin contained in the bispecific antibody is mutated at the 234th site, the 235th site and the 237th site according to the EU numbering system. The bispecific antibody of the application can specifically bind to CTLA4 and PD-1, specifically release the immune suppression of CTLA4 and PD-1 on the body, activate T lymphocytes, and has a good application prospect.

Description

[0001] This invention is a divisional application of the parent application with application number 202010757322.6, which was filed on July 31, 2020, and is entitled "Anti-CTLA4-Anti-PD-1 Bispecific Antibody and Its Use".

[0002] Cross-references to related applications

[0003] This application is based on and claims priority to CN application No. 201910711122.4 (filed on August 2, 2019) and CN application No. 201911224135.5 (filed on December 2, 2019), the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention belongs to the fields of tumor therapy and molecular immunology, and relates to an anti-CTLA4-anti-PD-1 bispecific antibody and its uses. Specifically, this invention relates to a mutated anti-CTLA4-anti-PD-1 bispecific antibody. Background Technology

[0005] The transmembrane receptor PD-1 (programmed cell death 1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (Programmed cell death 1 ligand 1) and PDL2 (Programmed cell death 1 ligand 2), both belong to the B7 superfamily. PDL1 is expressed in various cell types, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0006] The PD-1 / PDL1 signaling pathway plays a crucial role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is primarily expressed on immune cells such as T cells, while its ligand, PDL1, is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells, which then attack cancer cells. Blocking PD-1 / PDL1 signaling can also promote the proliferation of tumor antigen-specific T cells, which kill tumor cells and thus inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455–2465). Furthermore, tumors with high PDL1 expression are often associated with difficult-to-detect cancers (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA104:3360-5). One effective approach is to regulate PD-1 expression through in vivo injection of anti-PD-1 antibodies. Due to the broad-spectrum anti-tumor prospects and remarkable efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring about breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol. 42(3):466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets. 13(7):768-74).

[0007] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules share very similarities in gene structure, chromosomal location, sequence homology, and gene expression. Both are receptors for the co-stimulatory molecule B7 and are primarily expressed on the surface of activated T cells. CTLA4 binding to B7 inhibits the activation of mouse and human T cells, playing a negative regulatory role in T cell activation.

[0008] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing the responsiveness of T cells to various antigens. In vivo and in vitro studies have yielded largely consistent results in this regard. Currently, CTLA4 monoclonal antibodies are in clinical trials or have been approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, and malignant melanoma, among others (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun. 13:5.).

[0009] Interleukin-2 (IL-2), produced by T cells, is a growth factor regulating T cell subsets and an important factor in regulating immune responses. It also 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 the treatment of malignant tumors (including melanoma and renal tumors) and is currently undergoing clinical trials for the treatment of chronic viral infections (Chavez, AR, et al., 2009, Ann NY Acad Sci, 1182: p. 14-27). CTLA4 and CTLA4 antibodies, as important influencing factors of T cell function, intervene in the body's immune microenvironment. In in vitro and in vivo experiments, CTLA4 antibodies can specifically relieve the immunosuppression caused by CTLA4, activate T cells, and induce IL-2 production, showing broad application prospects in gene therapy for diseases such as tumors and parasites.

[0010] CTLA4 antibodies can produce specific therapeutic effects on diseases and exert high efficacy, supplementing the shortcomings of traditional drugs, thus opening up new avenues for gene therapy.

[0011] Bispecific antibodies, also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens. They can be produced through immunosorting purification. Alternatively, they can be obtained through genetic engineering. Genetic engineering methods offer advantages in terms of flexibility in binding site optimization, synthetic form considerations, and yield. Currently, more than 45 forms of bispecific antibodies have been proven to exist (Dafne Müller, Kontermann RE.2010, BioDrugs, 24(2):89-98). Currently, many bispecific antibodies have been developed in the form of IgG-scFv, i.e., the Morrison pattern (Coloma MJ, Morrison SL. 1997, Nat Biotechnol. 15:159-163). Due to its similarity to naturally occurring IgG, this form has advantages in antibody engineering, expression, and purification, and has been proven to be an ideal form of bifunctional antibody (Miller BR, Demarest SJ, et al., 2010, ProteinEng Des Sel; 23:549-57; Fitzgerald J, Lugovskoy A. 2011. MAbs; 3:299-309).

[0012] ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the Fab fragment of an antibody binding to the antigenic epitope of virus-infected cells or tumor cells, while its Fc fragment binds to the Fc receptor (FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0013] CDC (complement-dependent cytotoxicity) refers to the lysis of target cells caused by the specific binding of antibodies to corresponding antigens on the cell membrane, which forms a complex that activates the complement system. This process leads to the formation of MAC on the target cell surface. Complement can cause the lysis of various bacteria and other pathogenic cells, and is an important defense mechanism against pathogen infection.

[0014] Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells, used to recognize antibody Fc regions and mediate immune responses. After the antibody Fab region recognizes an antigen, its antibody Fc region binds to the Fc receptor on immune cells (such as killer cells), initiating immune cell responses such as phagocytosis and ADCC.

[0015] Based on the type of antibody recognized by the Fc receptor and the different cells expressing it, Fc receptors are mainly divided into three types: FcγR, FcαR, and FcεR. FcγR can be further divided into four subtypes: FcγRI (also known as CD64), FcγRII (also known as CD32), FcγRIII (also known as CD16), and FcRn (also known as Neonatal Fc receptor). Among them, FcγRI, FcγRII, and FcγRIII are closely related to ADCC effects. FcγRIII is the most important molecule mediating ADCC, and it has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. In the FcγRIIIa population, there are two subtypes: high-affinity FcγRIIIa caused by single nuclear stem polymorphism (SNP) sites, namely FcγRIIIa_V158 and low-affinity FcγRIIIa_F158. FcγRI has a high affinity for the Fc region of IgG and participates in the ADCC process. There are three subtypes of FcγRII: FcγRIIa, FcγRIIb, and FcγRIIc (also known as CD32a, CD32b, and CD32c, respectively). Among them, FcγRIIa has ADCC activity. There are two subtypes of FcγRIIa in the human population caused by single nucleotide mutations, namely FcγRIIa_H131 and FcγRIIa_R131. FcγRIIb is an inhibitory receptor. FcγRIIb is a typical inhibitory FcγR that can inhibit the nearby ITAM pathway. For example, after immune complexes bind 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).

[0016] The IgG family comprises four members: IgG1, IgG2, IgG3, and IgG4. The amino acid differences in the crystallizable (Fc) region of their heavy chain constant region result in varying affinities for FcγRs. IgG1 is the most abundant subtype in the human body and the most commonly used subtype in monoclonal antibody drugs. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the weakest affinity for FcγRs, but it can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 has the strongest binding affinity for FcγRs, inducing ADCC, and its CDC effect is stronger than that of IgG1. IgG4 molecules bind weakly to FcγRIs other than FcγRIs, and the likelihood of IgG4 molecules inducing CDC and NK cell-mediated ADCC is low. However, IgG4 subtype antibodies can mediate ADCP effects through binding to FcγRIs. The ADCP effect of antibody drugs targeting immune cells may lead to immune cell damage, resulting in negative effects on drug pharmacology. Currently, there is a need to develop novel anti-CTLA4-anti-PD-1 bispecific antibodies to reduce or eliminate antibody-mediated ADCC, ADCP, and / or CDC activity that could damage immune cells bound by anti-CTLA4-anti-PD-1 bispecific antibodies, thereby improving the efficacy of antibody drugs.

[0017] Chemotherapy drugs are currently mainly divided into the following nine categories (He Jie et al. Clinical Oncology. Beijing: People's Medical Publishing House, 2016: 230-237). The first category consists of drugs that directly bind to DNA and inhibit DNA replication, including various chemotherapeutic agents, mitomycin and bleomycin, dacarbazine, platinum-based drugs such as cisplatin and carboplatin, camptothecin-based drugs and their derivatives. The second category consists of drugs that inhibit nucleic acid biosynthesis. These drugs mainly affect the enzyme system of tumor cells, inhibiting the synthesis of DNA and RNA precursors, thereby inhibiting DNA or RNA formation. These drugs mainly include methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea, and cytarabine, etc. They mainly act on S phase cells and belong to antimetabolite chemotherapeutic drugs, which are cell cycle-specific anticancer drugs. The third category consists of chemotherapeutic drugs that affect transcription. Their main pharmacological action is to insert into the DNA double helix and form a non-covalent binding with it, thereby interfering with the transcription of genetic information on DNA into DNA-dependent mRNA, resulting in impaired signaling function and transcriptional inhibition. These drugs mainly include... The fourth category consists of drugs that affect microtubules and mitosis, mainly including vinblastines, podophyllotoxins, and paclitaxel-based herbal medicines. The fifth category consists of drugs that affect ribosome function and inhibit protein synthesis, represented by cephalotaxine-based herbal medicines. These drugs inhibit the initiation of protein synthesis, causing ribosome breakdown and the release of nascent peptide chains, but they cannot prevent the binding of mRNA and tRNA to ribosomes. These drugs can reduce nuclear DNA and cytoplasmic RNA, depolymerize polyribosomes, and inhibit mitosis. The sixth category consists of drugs that affect cell membranes, such as phytohemagglutinins (Con-A) and phytohemagglutinins (PHA). These can bind to glycoprotein receptors on the cell membrane, thereby affecting DNA synthesis in tumor cells and inhibiting tumor cell division. The seventh category consists of drugs that induce apoptosis. The first category includes drugs that regulate endocrine function, such as arsenic trioxide; the second category includes hormones that mainly treat tumors by regulating endocrine function, including estrogens, anti-estrogens, progestins, androgens, anti-androgens, adrenocortical hormones, and anti-adrenocortical hormones (including chlorobenzene dichloroethane and ammoniaglutide); the third category includes anti-tumor targeted therapy, including monoclonal antibodies, epidermal growth factor signaling inhibitors (such as targeted drugs against the receptor tyrosine kinase pathway), ubiquitin-proteasome inhibitors, and angiogenesis inhibitors.

[0018] Anlotinib is a quinoline derivative tyrosine kinase inhibitor that acts as a multi-target tyrosine kinase inhibitor (TKI) by influencing tumor angiogenesis and proliferation signaling. Its main targets include: receptor tyrosine kinases 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. A phase 2 trial showed that anlotinib improved progression-free survival and had a potential benefit in overall survival (Han B, et al. Br J Cancer. 2018; 118(5):654-661). A multicenter, double-blind, phase 3 randomized clinical trial showed that anlotinib led to prolonged overall survival and progression-free survival in Chinese patients. This finding suggests that anlotinib is well-tolerated 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).

[0019] Document WO2008112407 discloses a quinoline derivative tyrosine kinase inhibitor, 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinoline-7-yl]oxy]methyl]cyclopropylamine, and its preparation method in Example 24. Its structural formula is shown in Formula I below. Anlotinib hydrochloride is the hydrochloride salt of the compound of Formula I.

[0020]

[0021] Formula I

[0022] Lenvatinib is an oral multi-tyrosine kinase inhibitor developed by Eisai Co., Ltd. of Japan. It is a multi-target receptor tyrosine kinase inhibitor that inhibits the kinase activity of VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4). In addition to normal cellular function, lenvatinib also inhibits other receptor tyrosine kinases associated with pathogenic angiogenesis, tumor growth, and cancer progression, including fibroblast growth factor (FGF) receptors FGFR1, FGFR2, FGFR3, and FGFR4; transfection rearrangement receptor (RET), KIT, and platelet-derived growth factor receptor α (PDGFRα). Lenvatinib also exhibits antiproliferative activity in hepatocellular carcinoma cell lines, which depends on activated FGFR signaling and simultaneous inhibition of FGF receptor substrate 2α (FRS2α) phosphorylation.

[0023] The compound structure of lenvatinib is disclosed in US Patent 7,612,208, Example 368, as 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinoline carboxamide. US Patent No. 7,253,286 discloses the methanesulfonate form of lenvatinib (i.e., methanesulfonate), with the chemical name 4-[3-chloro-4-(cyclopropylureoyl)phenoxy]-7-methoxyquinoline-6-carboxamide methanesulfonate. Its chemical structure (Formula II) is provided below:

[0024] However, many cancer patients still cannot achieve long-term disease control after chemotherapy, and the 5-year survival rate remains very low. Therefore, developing less toxic and more effective treatments or combination therapy regimens has great clinical significance. Summary of the Invention

[0025] Through in-depth research and creative work, the inventors have modified the Fc terminus of the anti-CTLA4-anti-PD-1 antibody structure to reduce the binding of the Fc region to the Fc receptor, thereby reducing the toxic side effects of ADCC, ADCP and / or CDC on immune cells and increasing the efficacy of the anti-CTLA4-anti-PD-1 antibody drug.

[0026] This leads to the following invention: One aspect of the present invention relates to a bispecific antibody comprising: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin. in, The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 27-29, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 33-35, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38; or, The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 33-35 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 27-29 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32; The immunoglobulin is human IgG1 subtype; Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is mutated at any two or three sites among positions 234, 235 and 237, and after the mutation, the affinity constant of the bispecific antibody with FcγRIIIa and / or C1q is reduced compared with that before the mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction analyzer.

[0027] In one or more embodiments of the present invention, the bispecific antibody, after the above-mentioned mutation occurs, has a lower affinity constant compared with that of FcγRIIIa, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb before the mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction analyzer.

[0028] In one or more embodiments of the present invention, the bispecific antibody, according to the EU numbering system, has the following mutations in the heavy chain constant region of the immunoglobulin at positions 234, 235, and / or 237: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A.

[0029] In this invention, unless otherwise specified, the letter before the site represents the amino acid before the mutation, and the letter after the site represents the amino acid after the mutation.

[0030] This invention also relates to a bispecific antibody, comprising: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin. in, The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 27-29, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 33-35, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38; or, The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 33-35 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 27-29 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32; The immunoglobulin is human IgG1 subtype; Specifically, 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; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A.

[0031] In one or more embodiments of the present invention, the bispecific antibody, according to the EU numbering system, further comprises one or more mutations selected from the following: 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.

[0032] In one or more embodiments of the present invention, the bispecific antibody is in the form of IgG-scFv, i.e., Morrison mode.

[0033] In one or more embodiments of the present invention, the bispecific antibody, wherein... 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; and 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; and 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; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 4, SEQ ID NO: 8, 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; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO: 44; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 14 and SEQ ID NO: 18; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 16 and SEQ ID NO: 20.

[0034] In one or more embodiments of the present invention, the bispecific antibody is selected from any one of (1)-(20) below: (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4. (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8. (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 12. (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4. (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8. (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 12. (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16. (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20. (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 8; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16. (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 8; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20. (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16. (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20. (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 42. (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 44. (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 42. (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 44. (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 42; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16. (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 44; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16. (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 42; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20. as well as, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 44; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20.

[0035] In one or more embodiments of the present invention, the bispecific antibody: The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 40, and the amino acid sequence of its light chain is shown in SEQ ID NO: 24.

[0036] In one or more embodiments of the present invention, the bispecific antibody, wherein the immunoglobulin or its antigen-binding fragment has a concentration greater than about 10 -7 M, for example, greater than approximately 10 -6 M, 10 -5 M, 10 -4 M or 10 -3 An affinity constant of M or greater is combined with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the affinity constant is determined by a Fortebio Octet molecular interaction analyzer. Preferably, the immunoglobulin or its antigen-binding fragment has no binding signal or a binding signal less than 0.1 nm with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

[0037] In one or more embodiments of the present invention, the bispecific antibody, wherein the immunoglobulin or its antigen-binding fragment has a concentration greater than about 10 -9 M, for example, greater than approximately 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 An affinity constant of M or greater is used to bind C1q; preferably, the affinity constant is measured using a Fortebio Octet molecular interaction analyzer; Preferably, the immunoglobulin or its antigen-binding fragment has no binding signal with C1q or the binding signal is less than 0.1 nm; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

[0038] In one or more embodiments of the present invention, the bispecific antibody wherein the first protein functional region is directly linked to the second protein functional region or linked through a linker fragment; and / or the heavy chain variable region of the single-chain antibody is directly linked to the light chain variable region of the single-chain antibody or linked through a linker fragment.

[0039] In one or more embodiments of the present invention, the bispecific antibody is wherein the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0040] In one or more embodiments of the present invention, the bispecific antibody wherein the first protein functional region and the second protein functional region are independently one, two, or more.

[0041] In one or more embodiments of the present invention, the bispecific antibody comprises one first protein functional region and two second protein functional regions.

[0042] In one or more embodiments of the present invention, the bispecific antibody is wherein the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since immunoglobulins consist of two heavy chains, one immunoglobulin molecule is linked to two single-chain antibody molecules. Preferably, the two single-chain antibody molecules are identical. Preferably, the single-chain antibody is linked to the C-terminus of the immunoglobulin heavy chain via an amide bond formed by the aforementioned linker fragment.

[0043] In one or more embodiments of the present invention, the constant regions of the immunoglobulins are humanized, for example, the heavy chain constant regions are all Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant regions are all Ig kappa chain C region, ACCESSION: P01834.

[0044] In one or more embodiments of the present invention, the bispecific antibody, wherein the bispecific antibody is at a concentration of less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D It binds to CTLA4 protein and / or PD-1 protein.

[0045] In one or more embodiments of the present invention, the bispecific antibody is a monoclonal antibody.

[0046] In one or more embodiments of the present invention, the bispecific antibody is a humanized antibody.

[0047] In one or more embodiments of the present invention, the bispecific antibody comprises: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; The first protein has one functional region, and the second protein has two functional regions; Wherein, 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 shown in SEQ ID NO: 40, and the amino acid sequence of its light chain is shown in SEQ ID NO: 24. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 44. The single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker fragment; the first linker fragment and the second linker fragment may be 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 both as shown in SEQ ID NO:26.

[0048] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes the bispecific antibody described in any one of the present invention.

[0049] The present invention also relates to a carrier comprising the isolated nucleic acid molecules of the present invention.

[0050] The present invention also relates to a host cell containing the isolated nucleic acid molecules of the present invention, or the vector of the present invention.

[0051] Another aspect of the present invention relates to a conjugate comprising an antibody or an antigen-binding fragment thereof and a conjugation portion, wherein the immunoglobulin is a bispecific antibody as described in any one of the present invention, and the conjugation portion is a detectable label; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0052] Another aspect of the present invention relates to a kit comprising the bispecific antibody described in any one of the present invention, or comprising the conjugate of the present invention; Preferably, the kit further includes a second antibody that specifically recognizes the immunoglobulin or its antigen-binding fragment; optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0053] Another aspect of the invention relates to the use of any bispecific antibody or conjugate of the invention in the preparation of a kit for detecting the presence or level of PD-1 and / or CTLA4 in a sample.

[0054] Another aspect of the invention relates to a pharmaceutical composition comprising any one of the bispecific antibodies or conjugates of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0055] In one or more embodiments of the present invention, the pharmaceutical composition further comprises one or more tumor chemotherapy drugs; Preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate).

[0056] In one or more embodiments of the present invention, the pharmaceutical composition, wherein the unit dose of the pharmaceutical composition, calculated according to the mass of the bispecific antibody therein, is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0057] Another aspect of the invention relates to a combination product comprising individually packaged first and second products. in, The first product comprises the bispecific antibody, the conjugate of the present invention, or the pharmaceutical composition described in any one of the present invention. The second product comprises one or more tumor chemotherapy drugs; preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate). Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients. Preferably, the combined product further includes a product instruction manual.

[0058] In one or more embodiments of the present invention, the combined product, wherein the unit dose of the first product, calculated according to the mass of the bispecific antibody therein, is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0059] In one or more embodiments of the present invention, the combined product, wherein the unit dose of the second product, calculated according to the mass of the active ingredient therein, is 0.1mg-100mg, 0.5mg-50mg, 0.5mg-10mg, 1mg-10mg, 2mg-8mg, or 1mg-5mg.

[0060] In one or more embodiments of the present invention, the combined product, wherein the unit dose of the second product, calculated according to the mass of the active ingredient therein, is 1 mg-20 mg, 2 mg-15 mg, 4 mg-12 mg, or 8 mg-12 mg.

[0061] Another aspect of the present invention relates to the use of any bispecific antibody, conjugate, pharmaceutical composition, or combination product described in any one of the present invention in the preparation of medicaments for treating and / or preventing tumors or anemia, or in the preparation of medicaments for diagnosing tumors or anemia; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, and nasopharyngeal carcinoma; Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, it is colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumor with the 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 carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma.

[0062] MSI (microsatellite instability) refers to the instability of microsatellites. Microsatellites are short tandem repeats distributed throughout the human genome, consisting of single nucleotide, dinucleotide, or high-order nucleotide repeats, with repeat counts ranging from 10 to 50. Compared to normal cells, some abnormal tissue cells, such as tumors, exhibit altered microsatellite length due to the insertion or deletion of repeat units, a phenomenon known as MSI. Based on the degree of instability, MSI can be classified into highly unstable microsatellites (MSI-H), poorly unstable microsatellites (MSI-L), and stable microsatellites (MSS). The primary cause of MSI is a defect in DNA mismatch repair (MMR) function. Human mismatch repair genes (MMR genes) express corresponding mismatch repair proteins after transcription and translation. The absence of any MMR protein expression leads to a defect in the cell's mismatch repair function, resulting in the loss of repair function for base mismatches during DNA replication, causing accumulation and leading to microsatellite instability (MSI). Approximately 15% of colorectal cancers are caused by the MSI pathway. It was first discovered in colorectal cancer, but can also occur in gastric cancer, endometrial cancer, adrenocortical tumors, etc. (Baretti M et al. Pharmacol Ther. 2018;189:45‐62.). Subsequent studies have also found the MSI-H / dMMR feature in mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell tumors.

[0063] MSI-H and dMMR represent results from two different detection methods. dMMR and MSI-H are biologically consistent and are referred to as MSI-H / dMMR or MSI-high / dMMR. MSI-L and MSS represent the phenotype of proficient mismatch repair (pMMR). The dMMR detection method involves immunohistochemical protein detection of four mismatch genes—MSH2, MLH1, MSH6, and PMS2—in tumor specimens (including surgical and biopsy specimens). If any one of these four proteins is absent, it is dMMR; if all four proteins are positive, the tumor is pMMR, meaning the mismatch repair function is intact. MSI detection involves pairing and comparing the lengths of repetitive DNA sequences (microsatellite sequences) from tumor cells and somatic cells. When using PCR to detect five standard loci based on the US NCI standards, if two or more loci are inconsistent, it is considered unstable and defined as MSI-H; if one locus is inconsistent, it is called MSI-L (microsatellite low instability); and if all five loci are consistent, it is called MSS. High-throughput sequencing (or next-generation sequencing technology, NGS) can also be used to detect microsatellite instability. When using more microsatellite loci, such as more than five or other microsatellite loci, for PCR detection, ≥30% inconsistency is usually called MSI-H, all consistent loci are defined as MSS, and inconsistency between 0% and 30% is MSI-L.

[0064] Another aspect of the present invention relates to the use of any bispecific antibody or conjugate of the present invention in the preparation of the following pharmaceutical products: Drugs that block the binding of PD-1 and PDL1 Drugs that downregulate PD-1 activity or levels Drugs that relieve the immunosuppression caused by PD-1, or Increase IFN-γ in T lymphocytes and / or drugs that express IL-2; and / or Drugs that block the binding of CTLA4 and B7, Drugs that downregulate CTLA4 activity or CTLA4 levels Drugs that relieve the immunosuppression caused by CTLA4, or Drugs that increase IL-2 expression in T lymphocytes.

[0065] Another aspect of the present invention relates to a method for preventing and / or treating and / or adjuvant treating and / or diagnosing tumors, comprising the step of administering to a subject in need an effective amount of any one of the bispecific antibodies, conjugates, pharmaceutical compositions, or combination products of the present invention; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, and nasopharyngeal carcinoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

[0066] In one or more embodiments of the present invention, the method wherein the step of administering an effective amount of anti-CTLA4-anti-PD-1 bispecific antibody to the subject in need is performed before or after surgical treatment, and / or before or after radiotherapy.

[0067] In one or more embodiments of the present invention, the method wherein... The single-dose dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg); or, the single-dose dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 10-1000 mg per subject (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg or about 1000 mg), preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg; Preferably, the drug is administered once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the administration method is intravenous infusion or intravenous injection.

[0068] In some regimens, treatment with the anti-CTLA4-anti-PD-1 bispecific antibody is administered in cycles of 2 weeks (14 days) or 3 weeks (21 days), preferably intravenously on the first day (D1) of each cycle. For example, the anti-CTLA4-anti-PD-1 bispecific antibody is administered every two weeks (q2w) or every three weeks (q3w).

[0069] The bispecific antibody, conjugate, pharmaceutical composition, or combination product according to any one of the present invention is used for the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of tumors; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, and nasopharyngeal carcinoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

[0070] The bispecific antibody or conjugate according to any one of the present invention is used for: Blocking the binding of PD-1 to PDL1, Downregulate PD-1 activity or level, To relieve the body's immunosuppression caused by PD-1, or Increase IFN-γ in T lymphocytes Express; and / or Blocking the binding of CTLA4 to B7, Downregulate CTLA4 activity or CTLA4 levels To relieve the immunosuppression caused by CTLA4 in the body, or Increase IL-2 expression in T lymphocytes.

[0071] Antibody therapies, particularly monoclonal antibodies (mABs), have shown promising efficacy in treating a variety of diseases. Traditional experimental methods for obtaining these therapeutic antibodies involve immunizing animals with antigens to obtain antibodies targeting the antigens, or using affinity maturation methods to improve antibodies with low affinity for antigens.

[0072] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md).

[0073] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of the monoclonal antibody sequences in items (1)-(13) below through the VBASE2 database, the results are as follows: (1) 14C12 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0074] The amino acid sequences of the three CDR regions of its 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 its 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) (2) 14C12H1L1 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20.

[0075] The amino acid sequences of the three CDR regions in the heavy chain variable region are the same as those of 14C12.

[0076] The amino acid sequences of the three CDR regions in the light chain variable region are the same as those of 14C12.

[0077] (3) 4G10

[0078] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; The amino acid sequences of the three CDR regions of its 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 its 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) (4) 4G10H1L1 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. The amino acid sequences of the three CDR regions in the heavy chain variable region are the same as those of 4G10.

[0079] The amino acid sequences of the three CDR regions in the light chain variable region are the same as those of 4G10.

[0080] (5) 4G10H3L3

[0081] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12. The amino acid sequences of the three CDR regions in the heavy chain variable region are the same as those of 4G10.

[0082] The amino acid sequences of the three CDR regions in the light chain variable region are the same as those of 4G10.

[0083] (6) BiAb001(M)

[0084] The amino acid sequences of the nine CDR regions involved in its 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 involved in its 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) (7) BiAb002(M) The amino acid sequences of the nine CDR regions involved in its heavy chain variable region are identical to those of BiAb001(M).

[0085] The amino acid sequences of the three CDR regions involved in its light chain variable region are the same as those of BiAb001(M).

[0086] (8) BiAb003(M)

[0087] The amino acid sequences of the nine CDR regions involved in its heavy chain variable region are identical to those of BiAb001(M).

[0088] The amino acid sequences of the three CDR regions involved in its light chain variable region are the same as those of BiAb001(M).

[0089] (9) BiAb004(M)

[0090] The amino acid sequences of the nine CDR regions involved in its heavy chain variable region are identical to those of BiAb001(M).

[0091] The amino acid sequences of the three CDR regions involved in its light chain variable region are the same as those of BiAb001(M).

[0092] The antibody BiAb004 (hG1TM) of this invention introduces amino acid mutations in the non-variable region of BiAb004(M). Amino acid mutations are introduced at positions 234, 235, and 237 according to the EU numbering system. BiAb004 (hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in its heavy chain hinge region.

[0093] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0094] As used herein, when referring to the amino acid sequence of the CTLA4 protein (Cytotoxic T-Lymphocyte Antigen 4), it includes the full-length CTLA4 protein, or the extracellular fragment CTLA4ECD of CTLA4, or a fragment containing CTLA4ECD; it also includes fusion proteins of CTLA4ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in this invention, the term "CTLA4 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the CTLA4 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0095] As used herein, when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NM_005018), it includes the full-length PD-1 protein, or the extracellular fragment PD-1ECD of PD-1, or a fragment containing PD-1ECD; it also includes fusion proteins of PD-1ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the PD-1 protein without affecting its biological function. Therefore, in this invention, the term "added protein" should include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0096] As used herein, unless otherwise specified, B7 refers to B7-1 and / or B7-2; the specific protein sequences are known sequences in the art and can be found in existing literature or in GenBank. For example, B7-1 (CD80, NCBI Gene ID: 941); B7-2 (CD86, NCBI Gene ID: 942).

[0097] As used in this article, the term EC 50 The half-maximal effect concentration refers to the concentration that produces a 50% maximum effect.

[0098] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in an antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "amino acid regions" of approximately 12 or more amino acids; heavy chains also contain "amino acid regions" of approximately 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C LIt consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each region or domain forms an antibody binding site. The allocation of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. In particular, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of the present invention, the heavy chain may be the C-terminus of the heavy chain of an IgG antibody linked to the scFv of another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited to any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0099] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as those used for intact antibodies.

[0100] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0101] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see US Patent 4,816,567).

[0102] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, see, for example, Jones et al., Nature, 321:522 525 (1986); Reichmannet et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0103] As used herein, the term “epitope” refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. “Epitope” is also referred to in the art as an “antigenic determinant.” Epitopes or antigenic determinants typically consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, which can be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all points of interaction between the protein and interacting molecules (e.g., antibodies) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0104] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0105] As used herein, the term "Escherichia coli expression system" refers to an expression system consisting of Escherichia coli (strain) and a vector, wherein the Escherichia coli (strain) is derived from commercially available strains, such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR(DE3).

[0106] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0107] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as 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, HEK 293 cells, or human cells.

[0108] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D The antigen is bound to the antigen. In some embodiments of the invention, the term "targeted" refers to specific binding.

[0109] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have a dissociation equilibrium constant of less than approximately 10. -5M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D The antigen is bound, for example, by measuring it in a BIACORE instrument using surface plasmon resonance (SPR) or by measuring it using a Fortebio Octet molecular interaction instrument.

[0110] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0111] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means 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, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0112] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0113] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., diseases associated with CTLA4-B7 binding or excessive CTLA4 activity, such as cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., diseases associated with CTLA4-B7 binding or excessive CTLA4 activity, such as cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0114] "Recurrent" cancer is cancer that recurs at the initial site or a distant site after a response to initial treatment (such as surgery). "Locally recurrent" cancer is cancer that recurs at the same location as the previously treated cancer after treatment.

[0115] Metastatic cancer refers to cancer that has spread from one part of the body (such as the lungs) to another part of the body.

[0116] As used herein, the term "complete elimination" refers to the absence of binding signal or extremely low binding signal detected by existing instrumentation (e.g., the FortebioOctet molecular interaction analyzer). In one embodiment of the invention, absence of binding signal or extremely low binding signal means a binding signal (i.e., response value) below 0.1 nm.

[0117] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region, first linker fragment, or first product) and "second" (e.g., second protein functional region, second linker fragment, or second product) are used for distinguishing reference or clarifying expression, and do not have a typical sequential meaning.

[0118] In this invention, unless otherwise specified, “about” or “approximately” means fluctuating within 10%, 20% or 30% of the modified numerical value or physical quantity. For example, about 100 minutes or approximately 100 minutes can be 90-110 minutes, 80-120 minutes or 70-130 minutes.

[0119] Beneficial effects of the invention

[0120] The present invention achieves one or more of the technical effects described in items (1) to (3) below: (1) The inventors' modification of the Fc end of the antibody of the present invention completely eliminates the interaction between BiAb004(hG1TM) and Fc. C The binding activity of receptors FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIIa_F158 is eliminated, thereby eliminating ADCC and / or ADCP activity.

[0121] (2) The inventors completely eliminated the binding activity with complement C1q by modifying the Fc end of the antibody of the present invention, thereby completely eliminating CDC activity.

[0122] (3) The antibody of the present invention has the potential to be used in the preparation of drugs for the prevention and treatment of tumors. Attached Figure Description

[0123] Figure 1 Figure 1 shows the results of the affinity constant detection between BiAb004(hG1TM) and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0124] Figure 2 Figure 1 shows the affinity constant detection results of BiAb004(hG1WT)FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0125] Figure 3 Figure 1: Affinity constant detection results of BiAb004(hG1TM) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0126] Figure 4 Figure 1: Affinity constant detection results of BiAb004(hG1WT) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0127] Figure 5 Figure 1: Affinity constant detection results of BiAb004(hG1TM) and FcγRIIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0128] Figure 6Figure 1: Affinity constant detection results of BiAb004(hG1WT) and FcγRIIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0129] Figure 7 Figure 1: Affinity constant detection results of BiAb004(hG1TM) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0130] Figure 8 Figure 1: Affinity constant detection results of BiAb004 (hG1WT) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0131] Figure 9 Figure 1: Affinity constant detection results of BiAb004(hG1TM) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0132] Figure 10 Figure 1: Affinity constant detection results of BiAb004(hG1 WT) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0133] Figure 11 Figure 1: Affinity constant detection results of BiAb004(hG1TM) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0134] Figure 12 Figure 1: Affinity constant detection results of BiAb004(hG1WT) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0135] Figure 13 Figure 1 shows the results of the affinity constant detection between BiAb004(hG1TM) and C1q. The antibody concentrations added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0136] Figure 14 Figure 1 shows the results of the affinity constant detection between BiAb004(hG1WT) and C1q. The concentrations of antigen added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0137] Figure 15 Figure 1: Graph showing the affinity constants of 5C10H2L2-IgG1mt and C1q. The concentrations of antigen added to each pair of curves from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0138] Figure 16 Detection of ADCC activity in 293T-CTLA4-PD1 cells expressing CTLA-4 and PD-1 antigens by BiAb004(hG1WT) and BiAb004(hG1TM).

[0139] Figure 17 BiAb004 (hG1TM) in combination with lenvatinib inhibited the growth of subcutaneously inoculated human lung cancer HCC827 cells.

[0140] Figure 18 BiAb004 (hG1TM) inhibited the growth of subcutaneous xenografts of colon cancer MC38-hPDL1 / hCD73 in C57BL / 6-hPD1 / hPDL1 / hCD73 mice.

[0141] Figure 19 :BiAb004 (hG1TM) antibody-mediated phagocytic activity against CHO-K1-PD1.

[0142] Figure 20 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human gastric cancer cells KATO III.

[0143] Figure 21 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human cervical cancer cells (HeLa cells).

[0144] Figure 22 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human T-lymphoma cells, Jurkat cells.

[0145] Figure 23 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human nasopharyngeal carcinoma CNE-2Z cells.

[0146] Figure 24BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human breast cancer cells MDA-MB-231.

[0147] Figure 25 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to human skin tumor cells NCI-H2452.

[0148] Figure 26 BiAb004 (hG1TM) in combination with anlotinib significantly enhanced the immune response of immune cells against human non-small cell lung cancer cells (human lung adenocarcinoma cells) A549 cells.

[0149] Figure 27 BiAb004 (hG1TM) in combination with anlotinib significantly enhanced the immune response of immune cells against human small cell lung cancer cells NCI-H446.

[0150] Figure 28 BiAb004 (hG1TM) in combination with anlotinib significantly enhanced the immune response of immune cells against human lung squamous cell carcinoma NCI-H226 cells.

[0151] Figure 29 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to SW48 human colorectal cancer cells with the MSI-h / dMMR phenotype.

[0152] Figure 30 BiAb004 (hG1TM) significantly enhances the immune response of immune cells to SW837 human colorectal cancer cells with a non-MSI-h / dMMR phenotype. Detailed Implementation

[0153] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially.

[0154] In the following embodiments of the present invention: BALB / c mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0155] All human peripheral blood mononuclear cells were isolated and prepared at Zhongshan Kangfang Biopharmaceutical Co., Ltd., with informed consent from the provider.

[0156] Raji-PDL1 is a cell expressing human PD-L1 constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. based on the human B cell line Raji cells through transfection.

[0157] Ficoll-Paque TM PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.

[0158] The Human IL-2 ELISA Kit was purchased from Dakota Biotechnology Co., Ltd.

[0159] RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%), phenol red, and Blastidin were all derived from Gibco.

[0160] Staphylococcal enterotoxin antigen (SEB) is derived from Denotec.

[0161] FBS is derived from Excel bio.

[0162] Mitomycin C (MMC) is derived from Stressmarq.

[0163] The isotype control antibody is human anti-Hen Egg Lysozyme IgG (i.e., anti-HEL antibody, or human IgG, abbreviated as hIgG). The sequence of hIgG comes from the variable region of the Fab F10.6.6 sequence in Affinitymaturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-146.).

[0164] The anlotinib used in the examples is anlotinib hydrochloride, trade name Fucov®, generic name anlotinib hydrochloride, sourced from Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

[0165] The lenvatinib used in the examples is lenvatinib mesylate, marketed as Lenvatinib®, and sourced from Eisai (China) Pharmaceutical Co., Ltd.

[0166] Preparation Example 1: Sequence Design of Anti-CTLA4 Antibody

[0167] The heavy and light chain amino acid sequences and the encoding nucleic acid sequences of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1, and 4G10H3L3 in Chinese Patent Publication CN 106967172A.

[0168] (1) Heavy chain variable region sequence and light chain variable region sequence of 4G10

[0169] Nucleic acid sequence of the heavy chain variable region: (372 bp)

[0170] CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAA GTTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO: 1)

[0171] Its encoded amino acid sequence is: (124 aa)

[0172] QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2)

[0173] Nucleic acid sequence of the light chain variable region: (378 bp)

[0174] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATT CTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (SEQ ID NO: 3)

[0175] Its encoded amino acid sequence is: (126 aa)

[0176] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4)

[0177] (2) Heavy chain variable region sequence and light chain variable region sequence of humanized monoclonal antibody 4G10H1L1

[0178] Nucleic acid sequence of the heavy chain variable region (4G10H1V): (345 bp)

[0179] CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACCAACTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT (SEQ ID NO: 5)

[0180] The encoded amino acid sequence: (115 aa)

[0181] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 6)

[0182] Nucleic acid sequence of the light chain variable region (4G10L1V): (327 bp)

[0183] CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAATCGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG (SEQ ID NO: 7)

[0184] Its encoded amino acid sequence is: (109 aa)

[0185] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 8)

[0186] (3) Heavy chain variable region sequence and light chain variable region sequence of humanized monoclonal antibody 4G10H3L3

[0187] Nucleic acid sequence of the heavy chain variable region (4G10H3V): (345 bp)

[0188] CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACT AACTACGCACAGAAGTTCCAGGGGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (SEQ ID NO: 9)

[0189] Its encoded amino acid sequence (4G10H3V): (115 aa)

[0190] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 10)

[0191] Nucleic acid sequence of the light chain variable region (4G10L3V): (327 bp)

[0192] CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAAC AAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (SEQ ID NO: 11)

[0193] Its encoded amino acid sequence (4G10L3V): (109 aa)

[0194] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 12)

[0195] Preparation Example 2: Sequence Design of Anti-PD-1 Antibody 14C12 and its Humanized Antibody 14C12H1L1

[0196] The heavy and light chain amino acid sequences of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences, are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A.

[0197] (1) 14C12 heavy chain variable region sequence and light chain variable region sequence

[0198] Nucleic acid sequence of the heavy chain variable region: (354 bp)

[0199] GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 13)

[0200] The encoded amino acid sequence: (118 aa)

[0201] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 14)

[0202] The nucleic acid sequence of the light chain variable region: (321 bp)

[0203] GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 15)

[0204] Its encoded amino acid sequence is: (107 aa)

[0205] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 16)

[0206] (2) Heavy chain variable region sequence and light chain variable region sequence, heavy chain sequence and light chain sequence of humanized monoclonal antibody 14C12H1L1

[0207] Nucleic acid sequence of the heavy chain variable region: (354 bp)

[0208] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTAC TATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTTGCCTATTGGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 17)

[0209] Its encoded amino acid sequence is: (118 aa)

[0210] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18)

[0211] Nucleic acid sequence of the light chain variable region: (321 bp)

[0212] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 19)

[0213] The encoded amino acid sequence: (107 aa)

[0214] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 20)

[0215] The DNA sequence of the heavy chain of 14C12H1L1 (14C12H1): (1344 bp)

[0216]

[0217] Its encoded amino acid sequence: (448 aa)

[0218] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)

[0219] The DNA sequence of 14C12H1L1 light chain (the 14C12L1): (642 bp)

[0220] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 23)

[0221] The encoded amino acid sequence: (214 aa)

[0222] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24)

[0223] Preparation Example 3: Sequences of bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) design

[0224] The bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) have a Morrison pattern (IgG-scFv), in which the C-terminus of both heavy chains of an IgG antibody is linked to the scFv fragment of another antibody via a linker fragment. The design composition of their heavy and light chains is shown in Table A below.

[0225] Table A: Sequence design of BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M)

[0226] In Table A above:

[0227] The amino acid sequence of Linker1 is (GGGGS)3 (SEQ ID NO: 25).

[0228] The amino acid sequence of Linker2 is (GGGGS)4 (SEQ ID NO: 26).

[0229] In addition, in Table A above, the scFv fragments of the BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) antibodies, specifically 4G10H1V(M), 4G10L1V(M), 4G10H3V(M), and 4G10L3V(M), are derived from 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V respectively. These fragments involve mutations in individual amino acids in their backbone regions, effectively optimizing the antibody structure and improving its efficacy.

[0230] (1) 4G10H1V(M): (115 aa, amino acid sequence mutation sites based on 4G10H1V are underlined)

[0231] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 41)

[0232] (2) 4G10L1V(M): (110 aa, amino acid sequence mutation sites based on 4G10L1V are underlined)

[0233] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 42)

[0234] (3) 4G10H3V(M): (115 aa, amino acid sequence mutation sites based on 4G10H3V are underlined)

[0235] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 43)

[0236] (4) 4G10L3V(M): (110 aa, amino acid sequence mutation sites based on 4G10L3V are underlined)

[0237] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 44)

[0238] To distinguish it from the mutated antibody described below, BiAb004(M) in this embodiment of the invention is also referred to as BiAb004(hG1WT). The aforementioned BiAb004(M) is a "wild type" and uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0239] Preparation Example 4: Design of amino acid mutations in the non-variable region of the humanized bifunctional antibody BiAb004

[0240] Based on the BiAb004(hG1WT) obtained in Preparation Example 3, the inventors introduced 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 its heavy chain, thus obtaining BiAb004(hG1TM).

[0241] DNA sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1TM): (1344 bp, mutation sites are underlined)

[0242] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCT ACTATCCTGACAGCGTCAAGGCCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTTGCCTATTGGGGGGCAGGGAACCCTGGTGACAGTCTCT AGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGC AAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA (SEQ ID NO: 39)

[0243] Amino acid sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1TM): (448 aa, mutation sites are underlined)

[0244] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0245] The light chains of BiAb004(hG1TM) and BiAb004(hG1WT) have the same DNA sequence and encode the same amino acid sequence, as shown in Preparation Example 3.

[0246] Experimental Example 1: Detection of FcγRI affinity of BiAb004(hG1WT) and BiAb004(hG1TM) receptors

[0247] The Fc receptor FcγRI, also known as (CD64), can bind to the Fc terminus of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to the Fc receptor affects the safety and efficacy of the antibody.

[0248] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRI were detected using a Fortebio Octet molecular interaction analyzer to evaluate the potential ADCC and ADCP activities of each antibody.

[0249] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRI using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. FcγRI (purchased from Sinobio) solution at a concentration of 1 μg / mL was added to the HIS1K sensor and immobilized for 50 s to allow FcγRI to adhere to the sensor surface. The binding and dissociation parameters of the antibody with FcγRI were measured in buffer solution at antibody concentrations ranging from 3.12 to 50 nM (two-fold serial dilution). After the sensor immobilized with antigen was equilibrated in buffer solution for 60 s, the binding time of FcγRI immobilized on the sensor with each antibody was measured at 120 s; the dissociation time of FcγRI from the antibody was also measured at 120 s. The detection temperature was 30 °C, and the frequency was 0.3 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants of each antibody with FcγRI.

[0250] The results of affinity constant determination of FcγRI with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 1 below. Figure 1 , Figure 2 As shown.

[0251] Table 1: Kinetic parameters of the binding of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRI

[0252] The results showed that BiAb004(hG1WT) could bind to FcγRI with an affinity constant of 5.92E-09M; however, no data were obtained for BiAb004(hG1TM) due to the lack of binding signal or extremely low signal with FcγRI, and it was assumed that it did not bind to FcγRI.

[0253] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) effectively eliminated its binding activity with FcγRI.

[0254] Experimental Example 2: Determination of the affinity constants of FcγRIIIa_V158 with BiAb004(hG1WT) and BiAb004(hG1TM)

[0255] The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc terminus of IgG antibodies, mediating the ADCC effect.

[0256] In the experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIIa_V158 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0257] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRIIIa_V158 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_V158 bound to the antibodies (31.25–500 nM, 2-fold dilution) for 60 s. The antibodies dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.5 for 5 s, repeated 4 times. The detection temperature was 30 °C, and the frequency was 0.3 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0258] The affinity constants of FcγRIIIa_V158 with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 2 below. Figure 3 , Figure 4 As shown.

[0259] Table 2: Kinetic parameters of BiAb004(hG1WT) and BiAb004(hG1TM) combined with FcγRIIIa_V158

[0260] The results showed that BiAb004(hG1WT) could bind to FcγRI_V158 with an affinity constant of 1.77E-07M; however, BiAb004(hG1TM) did not bind to FcγRIIIa_V158 because there was no binding signal or the signal was extremely low, so no corresponding data could be obtained, and it was considered that it did not bind to FcγRIIIa_V158.

[0261] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) effectively eliminated its binding activity with FcγRIIIa_V158.

[0262] Experimental Example 3: Determination of the affinity constants of FcγRIIIa_F158 with BiAb004(hG1WT) and BiAb004(hG1TM)

[0263] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc terminus of IgG antibodies, mediating ADCC.

[0264] In this experiment, the affinity constants of BiAb004 (hG1WT) and BiAb004 (hG1TM) with FcγRIIIa_F158 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0265] The experimental method for detecting the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIIa_F158 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 5 μg / mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_F158 bound to the respective antibodies (31.25–500 nM, 2-fold dilution) for 60 s. The antibodies dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.5 for 5 s, repeated 4 times. The detection temperature was 30 °C, and the frequency was 0.3 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0266] The affinity constants of FcγRIIIa_F158 with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 3 below. Figure 5 , Figure 6 As shown.

[0267] Table 3: Kinetic parameters of BiAb004(hG1WT) and BiAb004(hG1TM) combined with FcγRIIIa_F158

[0268] The results showed that BiAb004(hG1WT) could bind to FcγRIIIa_F158 with an affinity constant of 2.21E-07M; however, BiAb004(hG1TM) did not bind to FcγRIIIa_F158 because there was no binding signal or the signal was too low to bind, so no data were obtained and it was assumed that it did not bind to FcγRIII_F158.

[0269] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) effectively eliminated its binding activity with FcγRIIIa_F158.

[0270] Experimental Example 4: Determination of the affinity constants of FcγRIIa_H131 with BiAb004(hG1WT) and BiAb004(hG1TM)

[0271] The Fc receptor FcγRIIa_H131, also known as (CD32a_H131), can bind to the Fc terminus of IgG antibodies, mediating ADCC.

[0272] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIa_H131 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0273] The experimental method for detecting the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIa_H131 using the Fortebio Octet molecular interaction analyzer is briefly described below: The immobilized material dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated for 300 s in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer to block the sensor. The FcγRIIa_H131 immobilized on the sensor bound to the antibody at a concentration of 12.5 μg / mL. The antibody was serially diluted 200 nM for 60 s, followed by dissociation in buffer for 60 s. The sensor was regenerated using 10 mM Glycine at pH 1.7 and 10 mM nickel sulfate. Detection was performed at 30 °C and 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0274] The affinity constants of FcγRIIa_H131 with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 4 below. Figure 7 , Figure 8 As shown.

[0275] Table 4: Kinetic parameters of BiAb004(hG1WT) and BiAb004(hG1TM) combined with FcγRIIa_H131

[0276] The results showed that BiAb004(hG1WT) could bind to FcγRIIa_H131 with an affinity constant of 2.22E-08M; however, BiAb004(hG1TM) did not bind to FcγRIIa_H131 because there was no binding signal or the signal was extremely low, so no corresponding data could be obtained, and it was considered that it did not bind to FcγRIIa_H131.

[0277] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) effectively eliminated its binding activity with FcγRIIa_H131.

[0278] Experimental Example 5: Determination of the affinity constants of FcγRIIa_R131 with BiAb004(hG1WT) and BiAb004(hG1TM)

[0279] The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) can bind to the Fc terminus of IgG antibodies, mediating ADCC.

[0280] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIa_R131 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0281] The experimental method for detecting the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) using the Fortebio Octet molecular interaction analyzer is briefly described below: The solidified material dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIa_R131 was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated for 300 s in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer to block the sensor. The FcγRIIa_R131 immobilized on the sensor bound to the antibody at a concentration of 12.5 μg / mL. The antibody was serially diluted 200 nM for 60 s, followed by dissociation in buffer for 60 s. The sensor was regenerated using 10 mM Glycine at pH 1.7 and 10 mM nickel sulfate. Detection was performed at 30 °C and 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0282] The affinity constants of FcγRIIa_R131 with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 5 below. Figure 9 , Figure 10 As shown.

[0283] Table 5: BiAb004(hG1WT) and BiAb004(hG1TM)

[0284] Dynamic parameters combined with FcγRIIa_R131

[0285] The results showed that both BiAb004(hG1WT) and BiAb004(hG1TM) could bind to FcγRIIa_R131, with affinity constants of 1.43E-08M and 4.20E-08M, respectively.

[0286] The results showed that both BiAb004(hG1WT) and BiAb004(hG1TM) exhibited binding activity with FcγRIIa_R131. However, Figure 9 and Figure 10 The results showed that BiAb004(hG1WT) had a higher binding signal and stronger affinity than BiAb004(hG1TM).

[0287] Experimental Example 6: Determination of the affinity constants of FcγRIIb with BiAb004(hG1WT) and BiAb004(hG1TM)

[0288] The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc terminus of IgG antibodies, negatively regulating the function of immune cells, inhibiting the activation and proliferation of immune cells, and inhibiting the secretion of cytokines.

[0289] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIb were detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of BiAb004(hG1WT) and BiAb004(hG1TM) to the Fc receptor.

[0290] The experimental method for detecting the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with FcγRIIb using the Fortebio Octet molecular interaction analyzer is briefly described below: The immobilized material dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIb was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated for 300 s in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer to block the sensor. The FcγRIIb immobilized on the sensor bound to the antibody at a concentration of 12.5–200 nM (two-fold serial dilution) for 60 s. The antibody then dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM Glycine, pH 1.7, and 10 mM nickel sulfate. The detection temperature was 30 °C, and the frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0291] The affinity constants of FcγRIIb with BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 6 below. Figure 11 , Figure 12 As shown.

[0292] Table 6: BiAb004(hG1WT) and BiAb004(hG1TM)

[0293] Kinetic parameters of FcγRIIb

[0294] The results showed that BiAb004(hG1WT) could bind to FcγRIIb with an affinity constant of 5.61E-08M; however, BiAb004(hG1TM) did not bind to FcγRIIb because there was no binding signal or the signal was extremely low, so no data were obtained, and it was assumed that it did not bind to FcγRIIb.

[0295] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) effectively eliminated its binding activity with FcγRIIb.

[0296] Experimental Example 7: Determination of the affinity of C1q with BiAb004(hG1WT) and BiAb004(hG1TM)

[0297] Serum complement C1q can bind to the Fc terminus of IgG antibodies, mediating the CDC effect. The ability of therapeutic monoclonal antibodies to bind to C1q affects the safety and efficacy of the antibody.

[0298] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with C1q were detected using a Fortebio Octet molecular interaction analyzer to evaluate the CDC activity of each antibody. In the experimental examples, the anti-PDL1 antibody 5C10H2L2-IgG1mt was used as a control antibody, and its preparation method followed PCT disclosure WO2017148424A1.

[0299] The experimental method for detecting the affinity constant of the corresponding antibody with C1q using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 50 μg / mL antibody was immobilized on the FAB2G sensor at a height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor then bound to the antigen C1q at a concentration of 1.25–20 nM (two-fold serial dilution) for 60 s. The antigen and antibody then dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 s, repeated 4 times. The sample plate vibration rate was 1000 rpm, the detection temperature was 30°C, and the detection frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Fortebio Data Acquisition 7.0 and Fortebio DataAnalysis 7.0 were used for data acquisition and analysis.

[0300] The affinity constants of C1q with BiAb004(hG1TM), BiAb004(hG1WT), and 5C10H2L2-IgG1mt are shown in Table 7 below. Figure 13 , Figure 14 , Figure 15 As shown.

[0301] Table 7: Kinetic parameters of binding of BiAb004(hG1WT), BiAb004(hG1TM), and 5C10H2L2-IgG1mt to C1q

[0302] The results showed that BiAb004 (hG1WT) could bind to C1q with an affinity constant of 2.53E-09M; however, BiAb004 (hG1TM) did not have a binding signal with C1q or the signal was extremely low, so no corresponding data could be obtained, and it was assumed that it did not bind to C1q.

[0303] The results showed that, compared with BiAb004 (hG1WT), BiAb004 (hG1TM) effectively eliminated its binding activity with C1q.

[0304] Experimental Example 8: BiAb004 (hG1WT) and BiAb004 (hG1TM) on the expression of CTLA4 and PD-1 antigens in 293T- ADCC activity assay of CTLA4-PD1 cells

[0305] ADCC (Advanced Difference-Cellular Compression) refers to the direct killing of target cells by effector immune cells with cytotoxic activity, which recognize the Fc fragment of antibodies bound to target cell antigens via their surface-expressed Fc receptors (FcRs). To detect the ADCC effect of the bifunctional anti-CTLA4-anti-PD-1 antibodies BiAb004(hG1WT) and BiAb004(hG1TM), the inventors constructed a co-culture system of 293T-CTLA4-PD1 cells expressing PD-1 and CTLA4 antigens and primary PBMCs to assess the antibody ADCC activity.

[0306] The ADCC activity of 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens was detected using BiAb004(hG1WT) and BiAb004(hG1TM). The specific methods are as follows: Normal human peripheral blood mononuclear cells (PBMCs) were isolated according to the Ficoll peripheral blood mononuclear cell isolation procedure instructions. The isolated PBMCs were resuspended in RPMI-1640 complete medium, AO / PI stained and counted, and then frozen. The day before the experiment, PBMCs were resuscitated, cell counts and viability were recorded, and the cells were incubated overnight in a 37 °C, 5% CO2 incubator. On the day of the experiment, 293T-CTLA4-PD1 cells and PBMCs were collected, centrifuged at 800 rpm or 1200 rpm for 5 min, and then resuspended in RPMI-1640 (containing 1% FBS) (hereinafter referred to as culture medium), and washed twice. Cell counts and viability were recorded, and the cell concentration was adjusted to a suitable range with culture medium. According to the experimental design, 100 μl of 293T-CTLA4-PD1 cells (3.0E+04 / well) were added to a 96-well plate; 50 μl of antibody was added and pre-incubated at room temperature for 1 hour; 1 hour later, 50 μl of PBMCs (9.0E+05 / well) were added and mixed thoroughly; the cells were incubated in a 37 °C, 5% CO2 incubator for 4 hours. Centrifuge at 250xg for 5 min; carefully aspirate 100 μl of cell supernatant (avoiding cells at the bottom of the plate) into a new 96-well flat-bottomed microplate, add 100 μl of freshly prepared reaction solution to each well according to the Cytotoxicity Detection Kit instructions, and incubate at room temperature in the dark for 30 min. Measure OD values ​​at 490 nm and 650 nm, respectively. Calculate the ADCC percentage for each group according to the formula: ADCC (%) = (Experimental group - Negative control group) / (Maximum LDH release from target cells - Spontaneous LDH release from target cells) × 100%.

[0307] The ADCC activity of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens was expressed as ADCC percentage. The results are as follows: Figure 16 As shown.

[0308] The results showed that in the mixed culture system of PBMC and 293T-CTLA4-PD1, at the same dose level, the percentage of ADCC induced by BiAb004 (hG1TM) was significantly lower than that induced by BiAb004 (hG1WT).

[0309] The results showed that BiAb004(hG1TM) had no ADCC activity.

[0310] Experimental Example 9: BiAb004 (hG1TM) combined with lenvatinib inhibited the growth of mouse colorectal cancer MC38 cell xenografts.

[0311] The mouse MC38 cell line is a mouse rectal cancer cell line and has been proven to be an effective model for studying human MSI-high / dMMR tumors (Efremova M et al. Nat Commun. 2018;9(1):32.).

[0312] MC-38 cells (purchased from Shanghai Lurui Biotechnology Co., Ltd.) were collected and the cell concentration was adjusted to 5 million / ml. 200 μl of each cell was subcutaneously injected into the right lumbar region of PD-1 transgenic mice (purchased from Shanghai Nanfang Model Biotechnology Co., Ltd.). One 100 μl of each mouse was injected. 10 6 One cell line was inoculated into 12 mice. When the tumor volume reached 60-150 mm², the cells were... 3 At approximately 10:00 AM, mice were divided into groups based on tumor volume (each group had roughly the same average tumor volume, approximately 94 mm²). 3 Two groups were established: a model group and a BiAb004 (hG1TM) combined with lenvatinib group, with 6 mice in each group. The day of grouping was designated D0. Antibody drugs were administered intraperitoneally on D0, D4, D7, D10, D13, and D17, for a total of 6 times. Lenvatinib was administered by gavage daily for 20 consecutive days. Tumor volume was measured using calipers. After grouping, tumor size was measured twice weekly using calipers and calculated using the formula TV = 0.5 × ab. 2 Calculate the tumor volume, where a is the major axis of the tumor, b is the minor axis of the tumor, and TV is the tumor volume. See Table 8 for the detailed experimental protocol.

[0313] Table 8: Experimental Design

[0314] The isotype control antibody used in this experiment is human anti-Hen EggLysozyme IgG (anti-HEL, i.e. human IgG, abbreviated as hIgG), whose sequence is derived from the variable region sequence of Fab F10.6.6 in the paper "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" published by Acierno et al. (Acierno et al. J MolBiol. 2007; 374(1): 130-46).

[0315] Experimental results are as follows Figure 17 As shown.

[0316] The results showed that the combination of BiAb004(hG1TM) and lenvatinib significantly inhibited the growth of MC38 tumors; the combination of BiAb004(hG1TM) and lenvatinib was significantly effective against rectal and / or colon cancer with the MSI-high / dMMR phenotype.

[0317] Experimental Example 10: BiAb004 (hG1TM) in C57BL / 6-hPD1 / hPDL1 / hCD73 mice with colon cancer MC38- Pharmacodynamic evaluation in hPDL1 / hCD73 subcutaneous transplantation model

[0318] Female C57BL / 6-hPD1 / hPDL1 / hCD73 mice (purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.), 8 mice per group, each mouse was subcutaneously inoculated with colon cancer MC38-hPDL1 / hCD73 cells (purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.) in the right forelimb (2 10 6 (cells / 100 μL / animal). The day of vaccination is defined as day D0. Adjust the dosing volume according to body weight: 10 μL / g Mouse body weight (g). Isotype Control antibody (preparation method and source same as in Example 9) or BiAb004 (hG1TM) were administered intraperitoneally; twice weekly for 3 weeks, for a total of 6 administrations. Tumors were continuously measured throughout the experiment, and the tumor volume (mm) was recorded as follows: 3 ) = (Tumor growth) (Tumor width) 2 The volume is calculated using the formula ) / 2.

[0319] Experimental results are as follows Figure 18 As shown in Table 9 below.

[0320] Table 9: Experimental Design and Grouping

[0321] The results showed that, compared with the isotype control antibody group, the growth of tumor volume in the BiAb004 (hG1TM) group was inhibited, indicating that BiAb004 (hG1TM) significantly inhibited the proliferation of mouse MC38 cells and could effectively treat colon cancer and / or rectal cancer.

[0322] Experimental Example 11: Antibody-mediated cytotoxicity of CHO-K1-PD1 by BiAb004(hG1WT) and BiAb004(hG1TM) Study on phagocytic activity

[0323] Studies by 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.) have shown that the binding of the Fc fragment of antibodies targeting immune checkpoints such as PD-1 and CTLA-4 to the Fc receptor has a negative impact on antibody-mediated anticancer activity. This may be due to immune cell damage induced by Fc-dependent effector function, among which antibody-dependent phagocytosis (ADCP) is an important mechanism leading to immune cell damage.

[0324] To detect the ADCP activity of BiAb004 (hG1TM), murine macrophages were used as effector cells, and cell lines overexpressing the corresponding antigen were used as target cells to investigate the ADCP-mediated effect. First, femoral bone marrow from C57BL / 6 mice (purchased from Guangdong Provincial Medical Laboratory Animal Center) was aseptically harvested and lysed on ice for 5 min with erythrocyte lysis buffer. Lysis was terminated with DMEM complete medium (containing 10% FBS), followed by washing twice by centrifugation at 1000 rpm. The cell pellet was resuspended in 10 mL of DMEM complete medium, and macrophage colony stimulating factor (M-CSF) was added to a working concentration of 100 ng / mL. Cells were induced and cultured at 37℃ and 5% CO2 for 7 days, with half the medium replaced and M-CSF added on days 3 and 5. Cell induction was completed on day 7. After digestion with 0.25% trypsin, macrophages were collected, centrifuged at 170xg for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium and counted. The cell density was adjusted and the cells were aliquoted into sterile EP tubes for later use.

[0325] Log-phase CHO-K1-CTLA4-PD1 cells (a cell line based on CHO-K1 cells simultaneously overexpressing human CTLA4 and PD1 antigens, constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd.) were collected, centrifuged at 170xg for 5 min, washed with PBS, resuspended, counted, and viability was determined. Carboxyfluorescein diacetates uccinimidyl ester (CFSE) was diluted to 2.5 μM with PBS. The diluted CFSE cells were resuspended (staining density: 10 million cells / mL) and incubated in a cell culture incubator for 20 min. Staining was terminated by adding 6 mL of DMEM complete medium, centrifuged at 170xg for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of DMEM complete medium, incubated in an incubator for 10 min, and adjusted to the experimental density. The cells were named CHO-K1-PD1-CTLA4-CFSE. The test antibody was diluted with DMEM complete medium. Simultaneously, an isotype control antibody (anti-HEL antibody) and culture medium were designed as the isotype control group and blank control group, respectively. According to the experimental design, diluted antibody and CHO-K1-PD1-CTLA4-CFSE cells were added to corresponding 1.5 mL EP tubes already containing macrophages (final volume 100 μL, effector cell to target cell ratio 50,000:150,000, antibody working concentrations 50, 5, and 0.5 nM), resuspended and mixed, and incubated at 37°C for 2 h. 800 μL of PBS solution containing 1% bovine serum albumin (BSA) at room temperature was added to each tube. Centrifuge for 5 min, discard supernatant; wash once with 800 μL of 1% PBSA. Dilute APC anti-mouse / human CD11b antibody (Biolegend, catalog number: 101212) 400-fold with 1% PBSA and add 100 μL / sample to the corresponding sample, mix well, incubate on ice for 30 min, and then wash each sample with 800 μL of 1% PBSA at 1200 mL / min. Centrifuge for 5 min, wash once, and discard the supernatant. Add 200 μL of 1% PBSA to each tube to resuspend the cells, transfer to flow cytometry tubes, and analyze using a BDFACSCalibur flow cytometer. The macrophages in the flow cytometry system are APCs. + Positive results were obtained from macrophages that underwent phagocytosis, which were double-positive for both APC and CFSE. The ratio of double-positive cells to APC-positive cells was used as the phagocytosis rate, and this ratio was used to evaluate antibody-mediated ADCP activity.

[0326] Calculate the ADCP activity of each group using the following formula, expressed as P%:

[0327] The results are as follows Figure 19 As shown.

[0328] The results showed that, under the antibody validation system with phagocytic activity, BiAb004(hG1TM) had no significant difference in macrophage phagocytosis activity of CHO-K1-PD1-CTLA4 cells compared with BiAb004(hG1WT), indicating that BiAb004(hG1TM) had no ADCP activity.

[0329] The results show that the amino acid mutation introduced by BiAb004(hG1TM) can effectively eliminate its ADCP effect, achieving unexpected technical results.

[0330] Experimental Example 12: BiAb004 (hG1TM) significantly enhanced the immune response of immune cells to human gastric cancer cells KATO III. Epidemic response

[0331] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and KATO III cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were routinely cultured in DMEM + 10% FBS complete medium. PBMCs were revived and activated with 0.5 μg / mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL mitomycin C (MMC) for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 5 cells / well were seeded in a 96-well plate; logarithmic growth phase KATO III cells were collected. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0332] The results are as follows Figure 20 As shown.

[0333] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human gastric cancer cells KATO III cells, manifested by a significant increase in IL-2 secretion levels, and has the potential to treat gastric cancer.

[0334] Experimental Example 13: BiAb004 (hG1TM) significantly enhanced the immunity of immune cells against human cervical cancer cells (HeLa cells). reaction

[0335] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and HeLa cells (purchased from the Cell Center of the Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 5 cells / well were seeded in a 96-well plate; logarithmic growth phase HeLa cells were collected, with each HeLa cell being a 5-cell-well plate. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISAKIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0336] The results are as follows Figure 21 As shown.

[0337] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human cervical cancer cells HeLa cells, manifested by a significant increase in IL-2 secretion levels, and has the potential to treat cervical cancer.

[0338] Experimental Example 14: BiAb004 (hG1TM) significantly enhanced the immune cells' ability to target human T-lymphoma cells (Jurkat cells). immune response

[0339] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals according to the instructions of the Ficoll-Paque™ Plus reagent. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells and human T-lymphoma cells (Jurkat cells, purchased from the Cell Center of the Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMCs were counted at 1. 10⁵ cells / well were seeded in a 96-well plate; logarithmic growth phase Jurkat cells were collected. Jurkat cells were 1... 10 5 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0340] The results are as follows Figure 22 As shown.

[0341] Compared to BiAb004(hG1WT), BiAb004(hG1TM) not only eliminates ADCC, CDC, and ADCP, but also significantly enhances the immune response of immune cells to human T-lymphoma cells (Jurkat cells), manifested by a significant increase in IL-2 secretion levels. Compared to BiAb004(hG1WT), it has the same or even higher pharmacological activity at dose levels of 1.34 nM and 20 nM, and has the potential to treat lymphoma.

[0342] Experimental Example 15: BiAb004 (hG1TM) significantly enhanced the immune response of immune cells to human nasopharyngeal carcinoma CNE-2Z cells. Epidemic response

[0343] According to the separation liquid Ficoll-Paque TMThe Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and CNE-2Z cells (purchased from Guangzhou Genio Biotechnology Co., Ltd.) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were revived and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 10 cells / well were seeded in a 96-well plate; NCE-2Z cells in logarithmic growth phase were collected. NCE-2Z cells were 3... 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0344] The results are as follows Figure 23 As shown.

[0345] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human nasopharyngeal carcinoma cells CNE-2Z, as evidenced by a significant increase in IL-2 secretion levels, especially at the 20 nM dose level.

[0346] The results above show that BiAb004(hG1TM) effectively removes the effects of ADCC, CDC and ADCP, and has better or comparable pharmacological activity than BiAb004(hG1WT), indicating its potential for treating human nasopharyngeal carcinoma.

[0347] Experimental Example 16: BiAb004 (hG1TM) significantly enhanced the immune cell response to human breast cancer cells MDA-MB-231. Cellular immune response

[0348] According to the separation liquid Ficoll-Paque TMThe Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and MDA-MB-231 cells (purchased from Guangzhou Genio Biotechnology Co., Ltd.) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were revived and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 10 cells / well were seeded into 96-well plates; logarithmic growth phase MDA-MB-231 cells were collected, with MDA-MB-231 cells being 3... 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0349] The results are as follows Figure 24 As shown.

[0350] Compared to BiAb004(hG1WT), BiAb004(hG1TM) enhanced the immune response of immune cells to human breast cancer MDA-MB-231 cells by an equal or more significant amount, as evidenced by a significant increase in IL-2 secretion levels.

[0351] The results above show that BiAb004(hG1TM) effectively removes the effects of ADCC, CDC and ADCP, and has better or comparable pharmacological activity than BiAb004(hG1WT), indicating its potential for treating human breast cancer.

[0352] Experimental Example 17: BiAb004 (hG1TM) significantly enhanced the immune cell response against human skin tumor cells NCI-H2452. Immune response

[0353] According to the separation liquid Ficoll-Paque TMThe Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and NCI-H2452 cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were revived and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 10 cells / well were seeded into 96-well plates; NCI-H2452 cells in logarithmic growth phase were collected. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0354] The results are as follows Figure 25 As shown.

[0355] The results showed that, compared with BiAb004(hG1WT), BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human mesothelioma cells NCI-H2452, as evidenced by a significant increase in IL-2 secretion levels, and has the potential to treat mesothelioma.

[0356] Experimental Example 18: BiAb004 (hG1TM) combined with anlotinib significantly enhanced the immune cells' response to human non-small cell lung cancer. Immune response of A549 cells (human lung adenocarcinoma cells)

[0357] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, and A549 cells were routinely cultured in DMEM + 10% FBS complete medium. PBMCs were resuscitated and activated with 0.5 μg / mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 55 cells / well were seeded into 96-well plates; logarithmic growth phase A549 cells (derived from the Cell Center of the Chinese Academy of Sciences) were collected. A549 cells were 5 cells / well. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody and anlotinib were added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0358] The results are as follows Figure 26 As shown, compared with BiAb004(hG1TM) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) combined with anlotinib hydrochloride, BiAb004(hG1TM) combined with anlotinib hydrochloride significantly enhanced the immune response of immune cells to human non-small cell lung cancer (human lung adenocarcinoma) A549 cells, as manifested by a significant increase in IL-2 secretion levels, showing potential for the treatment of human non-small cell lung cancer or human lung adenocarcinoma.

[0359] Experimental Example 19: BiAb004 (hG1TM) combined with anlotinib significantly enhanced the immune cells' response to human small cell lung cancer cells. Immune response of NCI-H446 cells

[0360] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and NCI-H446 cells (derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were revived and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 8 cells / well were seeded into 96-well plates; logarithmic growth phase NCI-H446 cells (derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were collected. NCI-H446 cells were 8 cells / well. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody and anlotinib were added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0361] The results are as follows Figure 27 As shown.

[0362] Compared with BiAb004(hG1TM) monotherapy, BiAb004(hG1WT) monotherapy, and BiAb004(hG1WT) in combination with anlotinib hydrochloride, BiAb004(hG1TM) in combination with anlotinib hydrochloride significantly enhanced the immune response of immune cells to human small cell lung cancer cells NCI-H446 cells, as evidenced by a significant increase in IL-2 secretion levels, demonstrating potential for the treatment of human small cell lung cancer.

[0363] Experimental Example 20: BiAb004 (hG1TM) combined with anlotinib significantly enhanced the effect of immune cells on human lung squamous cell carcinoma cells. Immunological response of NCI-H226 cells

[0364] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and NCI-H226 cells (derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1 × 10⁻⁶. 5 5 cells / well were seeded into 96-well plates; NCI-H226 cells in logarithmic growth phase were collected. NCI-H226 cells were seeded at 5 cells / well. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody and anlotinib were added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA kit instructions. The experimental medium was 10% FBS + RPMI 1640.

[0365] The results are as follows Figure 28 As shown.

[0366] Compared to BiAb004(hG1WT) monotherapy, BiAb004(hG1TM) monotherapy more effectively and significantly enhanced the immune response of immune cells to human lung squamous cell carcinoma cells NCI-H226 cells, as evidenced by a significant increase in IL-2 secretion levels.

[0367] Furthermore, compared to BiAb004(hG1WT) monotherapy and BiAb004(hG1TM) monotherapy, BiAb004(hG1TM) in combination with anlotinib hydrochloride significantly enhanced the immune response of immune cells to human lung squamous cell carcinoma cells NCI-H226 cells, and its pharmacological activity was comparable to that of BiAb004(hG1WT) in combination with anlotinib hydrochloride.

[0368] The results above show that BiAb004(hG1TM) effectively removes the effects of ADCC, CDC and ADCP, and has better or comparable pharmacological activity compared with BiAb004(hG1WT) monotherapy or BiAb004(hG1WT) in combination with anlotinib, indicating that it has a better therapeutic effect on human squamous cell carcinoma of the lung.

[0369] Experimental Example 21: BiAb004 (hG1TM) significantly enhanced immune cell activity against human colorectal cancer with the MSI-h / dMMR phenotype. Immune response of SW48 cells

[0370] SW48 cells, a human rectal cancer cell line, were identified as cells with the MSI-h / dMMR phenotype (Branch P et al. (1995). Cancer Res 55 (11): 2304–2309.) and were used to detect the immune response of BiAb004 (hG1TM)-enhanced immune cells to tumors with the MSI-h / dMMR phenotype.

[0371] In the experiment, the separation solution Ficoll-Paque was used. TM The Plus reagent instructions were followed to isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, and SW48 cells (from Guangzhou Genio Biotechnology Co., Ltd.) were routinely cultured in DMEM + 10% FBS complete medium. PBMCs were resuscitated and activated with 0.5 μg / mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 SW48 cells were seeded per well in a 96-well plate; logarithmic growth phase SW48 cells were collected, with SW48 cells being 2... 10 5Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0372] The results are as follows Figure 29 As shown.

[0373] Compared to anti-HEL antibodies, both BiAb004(hG1WT) and BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human colorectal cancer cells SW48 with the MSI-h / dMMR phenotype, as evidenced by a significant increase in IL-2 secretion levels.

[0374] The above results show that BiAb004(hG1TM) effectively removes the effects of ADCC, CDC and ADCP, and has better or comparable pharmacological activity compared with BiAb004(hG1WT), indicating its potential to treat solid tumors with the MSI-h / dMMR phenotype, especially colorectal cancer and / or rectal cancer with the MSI-h / dMMR phenotype.

[0375] Experimental Example 22: BiAb004 (hG1TM) significantly enhanced immune cell activity against the rectum of non-MSI-h / dMMR phenotyped humans. Immune response of cancer cells SW 837

[0376] SW 837 cells are human colorectal cancer cells with a non-MSI-h / dMMR (i.e. MSS) phenotype (Guo J et al. CancerRes. 2011;71(8):2978-2987.). In this example, they were used to detect the immune response of BiAb004 (hG1TM)-enhanced immune cells to tumors with a non-MSI-h / dMMR phenotype.

[0377] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals according to the instructions of the Ficoll-Paque™ Plus reagent. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, and SW837 cells (from Shanghai Hongshun Biotechnology Co., Ltd.) were routinely cultured in 10% FBS + Leibovitz's L-15 (from Gibco) complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1. 10 5 5 cells / well were seeded in a 96-well plate; logarithmic growth phase SW837 cells were collected, with 5 cells per well. 10 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions.

[0378] All experimental culture media were 10% FBS + RPMI 1640.

[0379] The results are as follows Figure 30 As shown.

[0380] Compared to anti-HEL antibodies, both BiAb004(hG1WT) and BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human colorectal cancer cells SW837 cells with non-MSI-h / dMMR phenotype. Among them, BiAb004(hG1TM) showed better pharmacological activity than BiAb004(hG1WT) in the high-dose group, as evidenced by a significant increase in IL-2 secretion levels.

[0381] The results above show that BiAb004(hG1TM) effectively removes the effects of ADCC, CDC, or ADCP, and has better or comparable pharmacological activity compared to BiAb004(hG1WT), indicating its potential to treat solid tumors with non-MSI-h / dMMR phenotypes, especially colorectal and / or rectal cancers with non-MSI-h / dMMR phenotypes.

[0382] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A bispecific antibody comprising: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; in, The immunoglobulin, wherein the heavy chain variable region comprises amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 27-29, and the light chain variable region comprises amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32; and the single-chain antibody, wherein the heavy chain variable region comprises amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 33-35, and the light chain variable region comprises amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38; The immunoglobulin is human IgG1 subtype; According to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutation: L234A, L235A, and G237A.

2. The bispecific antibody according to claim 1, wherein, According to the EU numbering system, the heavy chain constant region of the immunoglobulin also has one or more mutations selected from the following: 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.

3. The bispecific antibody according to any one of claims 1 to 2, wherein, 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; and 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; and 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; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO:

44.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the antibody is selected from any one of (1) to (10): （1） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

4. （2） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

8. （3） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

12. （4） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

4. （5） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

8. （6） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

12. （7） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

42. （8） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

44. （9） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

42. （10） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

44.

5. The bispecific antibody according to any one of claims 1 to 4, wherein: The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 40, and the amino acid sequence of its light chain is shown in SEQ ID NO:

24.

6. The bispecific antibody according to any one of claims 1 to 5, wherein, The immunoglobulin or its antigen-binding fragment is greater than 10 -7 M, for example, greater than approximately 10 -6 M, 10 -5 M, 10 -4 M or 10 -3 M or a larger dissociation equilibrium constant is combined with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the dissociation equilibrium constant is measured by a Fortebio Octet molecular interaction analyzer; Preferably, the immunoglobulin or its antigen-binding fragment has no binding signal or a binding signal less than 0.1 nm with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

7. The bispecific antibody according to any one of claims 1 to 6, wherein, The immunoglobulin or its antigen-binding fragment is greater than 10 -9 M, for example, greater than approximately 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 M or a larger dissociation equilibrium constant combined with C1q; preferably, the dissociation equilibrium constant is measured by a Fortebio Octet molecular interaction analyzer; Preferably, the immunoglobulin or its antigen-binding fragment has no binding signal with C1q or the binding signal is less than 0.1 nm; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

8. The bispecific antibody according to any one of claims 1 to 7, wherein, The first protein functional region is directly connected to the second protein functional region or connected through a linker fragment; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker fragment.

9. The bispecific antibody according to claim 8, wherein, The connecting segment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

10. The bispecific antibody according to any one of claims 1 to 9, wherein, The first protein functional region and the second protein functional region are independently one, two, or more.

11. The bispecific antibody according to any one of claims 1 to 10, wherein, The single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin.

12. A bispecific antibody comprising: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; The first protein has one functional region, and the second protein has two functional regions; Wherein, 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 shown in SEQ ID NO: 40, and the amino acid sequence of its light chain is shown in SEQ ID NO:

24. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

44. The single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker fragment; the first linker fragment and the second linker fragment may be 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 both as shown in SEQ ID NO:

26.

13. An isolated nucleic acid molecule encoding the bispecific antibody as described in any one of claims 1 to 12.

14. A carrier comprising the isolated nucleic acid molecule of claim 13.

15. A host cell comprising the isolated nucleic acid molecule of claim 13, or the vector of claim 14.

16. A conjugate comprising an antibody or an antigen-binding fragment thereof and a conjugated portion, wherein, The antibody is a bispecific antibody as described in any one of claims 1 to 12, and the conjugated portion is a detectable label; preferably, the conjugated portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

17. A kit comprising the bispecific antibody of any one of claims 1 to 12, or comprising the conjugate of claim 16; Preferably, the kit further includes a second antibody that specifically recognizes the immunoglobulin or its antigen-binding fragment; optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

18. Use of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 16 in the preparation of a kit for detecting the presence or level of PD-1 and / or CTLA4 in a sample.

19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 16; optionally, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier and / or excipient.

20. The pharmaceutical composition according to claim 19, wherein, The unit dose of the pharmaceutical composition, calculated according to the mass of the bispecific antibody therein, is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

21. Use of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 16 in the preparation of a medicament for treating and / or preventing tumors or anemia, or in the preparation of a medicament for diagnosing tumors or anemia; Preferably, the tumor is selected from one or more of melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, and nasopharyngeal carcinoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

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