Bispecific antibody targeting B7H3 and 4-1BB and application thereof

Abstract

The invention discloses a bispecific antibody targeting to B7H3 and 4-1BB and an application of the bispecific antibody. The invention provides a bispecific antibody targeting B7H3 and 4-1BB. The bispecific antibody is formed by connecting a 4-1BB antigen binding domain to C ends of two heavy chains of an anti-B7H3 antibody respectively; the 4-1BB antigen binding structural domain is a single-chain antibody for resisting 4-1BB, and comprises a heavy chain variable region for resisting the 4-1BB antibody and a light chain variable region for resisting the 4-1BB antibody. The bispecific antibody provided by the invention shows a good in-vitro tumor cell killing effect and an in-vivo tumor inhibition effect, so that the bispecific antibody provided by the invention has important significance and application potential when being used for preparing antibody targeted drugs.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment and biomedicine technology, and in particular to a bispecific antibody targeting B7H3 and 4-1BB and an application thereof. Background Art

[0002] Bispecific antibodies (BsAb), also known as bifunctional antibodies, can simultaneously recognize and bind to two different antigens or epitopes compared to ordinary antibodies that can only bind to a single target. They can play some special biological functions and are even better than the synergistic effect of two monoclonal antibodies combined. Therefore, they are increasingly becoming one of the new hotspots in antibody drug research.

[0003] B7-H3 (CD276 / B7RP-2), a member of the B7 ligand family (PD-L1 is also a member of this family), is overexpressed in most cancer types, but expressed at low levels in normal tissues. In malignant tissues, B7-H3 inhibits tumor antigen-specific immune responses and promotes tumor growth. B7-H3 also has non-immunogenic functions, such as promoting migration and invasion, angiogenesis, chemotherapy resistance, endothelial cell to mesenchymal cell transformation, and affecting tumor cell metabolism. The expression of B7-H3 in tumors is associated with poor prognosis, and more and more people are beginning to pay attention to the application value of this target in tumor immunotherapy.

[0004] The co-stimulatory molecule 4-1BB is an important member of the tumor necrosis factor receptor superfamily and is a co-stimulatory signal that mediates T cell activation. 4-1BB-mediated co-stimulatory signals can enhance the function of T cells and improve T cells' surveillance of tumor cells and immune defense against viral infections. Regulating the immune function of lymphocytes by intervening in the 4-1BB pathway may become a new immunotherapy approach. Summary of the invention

[0005] The purpose of the present invention is to provide a bispecific antibody targeting B7H3 and 4-1BB and its application. The bispecific antibody can simultaneously recognize two antigen targets, CD137, namely 4-1BB and B7H3.

[0006] The technical problem to be solved by the present invention is: how to avoid the toxic and side effects caused by the large-scale activation of the immune system during targeted therapy.

[0007] To solve the above technical problems, in the first aspect, the present invention claims a bispecific antibody targeting B7H3 and 4-1BB. This bispecific antibody includes an immune agonist module, such as a 4-1BB antigen binding domain, which can specifically bind to the 4-1BB antigen, and a tumor targeting module, such as a B7H3 antigen binding domain, which can specifically bind to a tumor-associated antigen.

[0008] The bispecific antibody targeting B7H3 and 4-1BB claimed in the present invention is specifically formed by connecting a 4-1BB antigen binding domain to the C-terminus of the two heavy chains of the anti-B7H3 antibody respectively.

[0009] Wherein, the 4-1BB antigen binding domain can be connected to the C-terminus of the heavy chain of the anti-B7H3 antibody through a connecting peptide. The connecting peptide can be composed of a positive integer multiple of GGGGS subunits, such as 1, 2, 3, 4, 5 or 6. Preferably, in the bispecific antibody, the connecting peptide is composed of 3 GGGGS subunits. Of course, the connecting peptide described here can also be other flexible peptides, such as A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, etc.

[0010] Wherein, the 4-1BB antigen binding domain is a single-chain antibody (scFv), i.e., only comprising the heavy chain variable region and light chain variable region of the antibody. In a specific embodiment of the present invention, the 4-1BB antigen binding domain is formed by sequentially connecting the light chain variable region of the anti-4-1BB antibody, the connecting peptide and the heavy chain variable region of the anti-4-1BB antibody. The connecting peptide can be composed of a positive integer multiple of GGGGS subunits, such as 1, 2, 3, 4, 5 or 6, and preferably, in a specific embodiment of the present invention, the connecting peptide is composed of 4 GGGGS subunits.

[0011] Furthermore, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-B7H3 antibody are shown in positions 31-35 of SEQ ID No.1, positions 47-66 of SEQ ID No.1, and positions 99-107 of SEQ ID No.1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-B7H3 antibody are shown in positions 24-34 of SEQ ID No.2, positions 50-56 of SEQ ID No.2, and positions 89-97 of SEQ ID No.2, respectively.

[0012] Furthermore, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-4-1BB antibody are shown in positions 620-624 of SEQ ID No.1, positions 639-654 of SEQ ID No.1, and positions 687-695 of SEQ ID No.1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-4-1BB antibody are shown in positions 486-496 of SEQ ID No.1, positions 512-518 of SEQ ID No.1, and positions 551-559 of SEQ ID No.1, respectively.

[0013] In the present invention, CDR is "complementarity determining region", which is a region in the variable domain of an antibody that is highly variable in sequence and forms a structurally determined "hypervariable loop" and / or contains antigen contact residues "antigen contact points". CDR is mainly responsible for binding to antigen epitopes. A variable region usually contains 3 CDR regions, CDR1, CDR2 and CDR3 from the N-terminus. HCDR1, HCDR2 and HCDR3 represent the 3 CDR regions in the heavy chain variable region, and LCDR1, LCDR2 and LCDR3 represent the 3 CDR regions in the light chain variable region.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-B7H3 antibody is shown as positions 1-118 of SEQ ID No.1; the amino acid sequence of the light chain variable region of the anti-B7H3 antibody is shown as positions 1-107 of SEQ ID No.2.

[0015] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-4-1BB antibody is shown as positions 590-706 of SEQ ID No.1; the amino acid sequence of the light chain variable region of the anti-4-1BB antibody is shown as positions 463-569 of SEQ ID No.1.

[0016] More specifically, the amino acid sequence of the 4-1BB antigen binding domain is shown in positions 463-706 of SEQ ID No.1.

[0017] Furthermore, the heavy chain constant region in the bispecific antibody may be selected from any one of the following:

[0018] 1), IgG, IgM, IgE, IgA or IgD;

[0019] 2), IgG1, IgG2, IgG3 or IgG4;

[0020] 3) IgG4 mutants with S228P, F234A and L235A mutations.

[0021] In one specific embodiment of the present invention, the heavy chain constant region in the bispecific antibody is IgG1 (corresponding to antibody HK056-001); in another specific embodiment of the present invention, the heavy chain constant region in the bispecific antibody is an IgG4 mutant with S228P, F234A and L235A mutations (corresponding to antibody HK056-002).

[0022] Furthermore, the bispecific antibody is composed of two heavy chains and two light chains. In a specific embodiment of the present invention, the amino acid sequence of one chain (heavy chain) of the bispecific antibody is shown in SEQ ID No.1, and the amino acid sequence of the other chain (light chain) is shown in SEQ ID No.2.

[0023] In a specific embodiment of the present invention, the anti-B7H3 antibody is from patent PCT / US / 2015 / 047013; the anti-4-1BB antibody is from patent WO2021093753A1.

[0024] In a second aspect, the present invention claims protection for substances related to the bispecific antibody described in the first aspect above.

[0025] The substance related to the bispecific antibody described in the first aspect above and claimed in the present invention may be any of the following:

[0026] (A1) a nucleic acid molecule encoding the bispecific antibody described in the first aspect above;

[0027] (A2) an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule described in (A1);

[0028] (A3) A pharmaceutical composition comprising the bispecific antibody described in the first aspect above.

[0029] Further, in (A1), the nucleotide sequence encoding the heavy chain variable region of the anti-4-1BB antibody is shown in positions 1768-2118 of SEQ ID No. 3; the nucleotide sequence encoding the light chain variable region of the anti-4-1BB antibody is shown in positions 1387-1707 of SEQ ID No. 3. The nucleotide sequence encoding the heavy chain variable region of the anti-B7H3 antibody is shown in positions 1-354 of SEQ ID No. 3; the nucleotide sequence encoding the light chain variable region of the anti-B7H3 antibody is shown in positions 1-321 of SEQ ID No. 4.

[0030] Furthermore, the nucleotide sequence encoding the 4-1BB antigen binding domain is shown in 1387-2118 of SEQ ID No.3.

[0031] More specifically, the nucleotide sequence encoding one chain of the bispecific antibody is shown in SEQ ID No.3, and the nucleotide sequence encoding the other chain of the bispecific antibody is shown in SEQ ID No.4.

[0032] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid A and / or a recombinant plasmid B. The recombinant plasmid A is a recombinant plasmid obtained by cloning the coding gene (SEQ ID No.3) of one chain of the bispecific antibody into the pcDNA3.4 vector (such as between the XbaI and HindIII sites). The recombinant plasmid B is a recombinant plasmid obtained by cloning the coding gene (SEQ ID No.4) of the other chain of the bispecific antibody into the pcDNA3.4 vector (such as between the XbaI and HindIII sites). Correspondingly, the recombinant cell line is a cell line containing the recombinant vector. In a specific embodiment of the present invention, the recombinant cell line is obtained by introducing the recombinant vector into the human embryonic kidney cell line HEK293F.

[0033] Further, in (A3), the pharmaceutical composition may also include targeting other immune checkpoints such as PD-1, TIM-3, LAG-3, CTLA-4, OX40, etc., or targeting other TAAs such as B7H3, VEGF, EGFR, PD-L1, etc., as well as small molecules such as peptides and chemical drugs, etc. In a specific embodiment of the present invention, the pharmaceutical composition consists of the bispecific antibody and an anti-PD-1 antibody.

[0034] Furthermore, in (A3), the pharmaceutical composition may further include a pharmaceutically acceptable excipient, diluent or carrier.

[0035] In a third aspect, the present invention claims a method for preparing the bispecific antibody described in the first aspect above.

[0036] The method for preparing the bispecific antibody described in the first aspect above as claimed in the present invention may include the following steps: introducing the nucleic acid molecule encoding the bispecific antibody described in the second aspect above into a recipient cell, and obtaining the bispecific antibody after culturing.

[0037] In the method, after the nucleic acid molecule encoding the bispecific antibody is translated into corresponding polypeptide chains in the recipient cell, the four polypeptide chains will self-assemble into the bispecific antibody of the present invention.

[0038] The method further includes: a step of separating and purifying the bispecific antibody by affinity chromatography, and the bispecific antibody can be purified into a substantially uniform substance by this method.

[0039] Furthermore, the nucleic acid molecule encoding the bispecific antibody can be introduced into the recipient cell in the form of a recombinant vector, wherein the recombinant vector can be the recombinant vector described in the second aspect above.

[0040] Furthermore, the receptor cell may be a mammalian cell or an insect cell, and the mammalian cell may be a human kidney epithelial cell line HEK293F.

[0041] In a fourth aspect, the present invention claims the use of the bispecific antibody described in the first aspect or the substance described in the second aspect in any of the following:

[0042] (B1) Preparation of antibody targeted drugs;

[0043] (B2) preparing a product for activating T cells;

[0044] (B3) preparing products that kill tumor cells;

[0045] (B4) Preparation of products for preventing and / or treating tumors.

[0046] Furthermore, the product may be an antibody drug, CAR-T, CAR-NK, antibody-drug conjugate, immunotoxin and vaccine, etc.

[0047] Furthermore, the activated T cells are activated in the presence of tumor cells expressing B7H3.

[0048] Furthermore, the killing of tumor cells is the bispecific antibody mediating PBMC killing of tumor cells, which may specifically include: mediating NK cells to kill tumor cells through ADCC effect, and mediating T cells to directly kill tumor cells.

[0049] Wherein, the tumor cells are tumor cells expressing B7H3; and the tumor is a tumor expressing B7H3.

[0050] Further, the tumor may be selected from the following: mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, bile duct cancer, endometrial cancer, thymic cancer, colon cancer and breast cancer.

[0051] In a specific embodiment of the present invention, the tumor cells are specifically selected from the following: breast cancer cells (such as MCF-7 cells), ovarian cancer cells (such as SKOV3 cells), prostate cancer cells (such as PC-3 cells), colon cancer cells (such as CT26 cells or MC38 cells transfected with B7H3).

[0052] In a specific embodiment of the present invention, the tumor is specifically selected from the following: breast cancer, ovarian cancer, prostate cancer, and colorectal cancer.

[0053] The beneficial technical effects achieved by the present invention are as follows:

[0054] 1. The bispecific antibody targeting B7H3 and 4-1BB claimed in the present invention can simultaneously recognize two antigen targets, 4-1BB and B7H3.

[0055] The bispecific antibody can bind to B7H3 and 4-1BB simultaneously.

[0056] The bispecific antibody can activate PBMC in in vitro experiments, specifically activate T cells in PBMC, and enhance the level of IFN-γ secretion.

[0057] The activation effect of the bispecific antibody on PBMCs is dependent on B7H3 and is inactive in the absence of B7H3.

[0058] The bispecific antibody can activate the 4-1BB signaling pathway, and this activation depends on crosslinking.

[0059] The bispecific antibody can enhance the killing of tumor cells by NK cells and PBMC in in vitro experiments.

[0060] 2. The bispecific antibody has anti-tumor efficacy, and the bispecific efficacy is better than that of the corresponding bispecific antibody without Fc function. The bispecific antibody has the ability to simultaneously recognize the tumor cell surface antigen B7H3 and the immune cell surface antigen 4-1BB, and activates immune cells by efficiently enriching B7H3 near tumor cells, which can effectively avoid the systemic toxicity caused by the activation of systemic immune cells by 4-1BB agonistic antibodies, and can induce adaptive immunity by binding to Fc receptors, thereby achieving efficient anti-tumor effects.

[0061] 3. Experiments have shown that the bispecific antibodies provided by the present invention have good T cell activation effects, and on this basis, they exhibit good in vitro tumor cell killing effects and in vivo tumor inhibition effects. Therefore, the bispecific antibodies provided by the present invention have important significance and application potential for the preparation of antibody targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the structure of a bispecific antibody molecule.

[0063] Figure 2 The electrophoresis (SDS-PAGE) results of the bispecific antibody under non-reducing conditions and reducing conditions.

[0064] Figure 3 Flow cytometry was used to detect the activity of the bispecific antibody molecule binding to the B7H3 antigen.

[0065] Figure 4 Flow cytometry was used to detect the activity of bispecific antibody molecules binding to 4-1BB antigen.

[0066] Figure 5Flow cytometry was used to detect the activity of the bispecific antibody molecule in simultaneously binding to human B7H3 antigen and human 4-1BB antigen.

[0067] Figure 6 B7H3-mediated bispecific antibody molecules activate PBMCs to produce IFN-γ.

[0068] Figure 7 The ADCC effect of bispecific antibody molecules on tumor cells was detected by reporter gene.

[0069] Figure 8 It is the ADCC killing effect of human PBMC on tumor cells mediated by bispecific antibody molecules.

[0070] Fig. 9 It is the direct killing effect of human PBMC on tumor cells mediated by bispecific antibody molecules.

[0071] Fig.10 The reporter gene was used to detect the activation of 4-1BB signaling pathway by B7H3-mediated bispecific antibody molecules.

[0072] Fig.11 To detect the inhibitory effect of bispecific antibody molecules on CT26 / HuB7H3 model tumor growth in vivo.

[0073] Fig.12 This is a challenge experiment for the CT26 / HuB7H3 tumor model.

[0074] Fig.13 To test the inhibitory effect of bispecific antibodies on MC38 / HuB7H3 tumor growth in vivo. DETAILED DESCRIPTION

[0075] Terms and Definitions

[0076] BsAb: bispecific antibody;

[0077] TAA: Tumor associated antibody;

[0078] VH: heavy chain variable domain;

[0079] VL: Light chain variable domain;

[0080] FACS: Fluorescence-activated cell sorting, also known as flow cytometry;

[0081] SDS-PAGE: polyacrylamide gel electrophoresis;

[0082] PBMC: peripheral blood mononuclear cells;

[0083] CR: The abbreviation of Complete release, which means the tumor has completely disappeared.

[0084] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0085] As used herein, when referring to the amino acid sequence of 4-1BB protein or 4-1BB protein (UniProt Q07011), it includes the full length of 4-1BB protein, or the extracellular fragment 4-1BB-ECD of 4-1BB or a fragment comprising 4-1BB-ECD; Also including the fusion protein of 4-1BB-ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. The term "4-1BB protein" includes all such sequences, including its natural or artificial variants.

[0086] As used herein, the term EC50 refers to concentration for 50% of maximal effect, which refers to the concentration that can induce 50% of the maximal effect.

[0087] As used herein, the term "monoclonal antibody" or Antibody, unless otherwise specified, generally refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains (each pair has a "light" (L) chain and a "heavy" (H) chain. In a general sense, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain refers to the polypeptide chain with a smaller molecular weight in the antibody. Light chains can be classified as κ and λ light chains. Heavy chains can usually be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH 1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate 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. The VH and VL regions can also be subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antibody binding sites, respectively. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain can also contain more than 3 CDRs, such as 6, 9, or 12. For example, in the bifunctional antibody of the present invention, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody connected to the scFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), an IgA1, IgA2, IgD, IgE or IgM antibody.

[0088] Antibody antigen-binding fragments (e.g., those 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 cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0089] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0090] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0091] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0092] 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.

[0093] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The term "targeting" refers to specific binding.

[0094] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0095] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0096] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0097] The following examples do not include a detailed description of conventional methods, such as those used for gene amplification, recombinant plasmid construction, and the introduction of plasmids into host cells. Such methods are described in many publications, including Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0098] pcDNA3.4 vector: Invitrogen, Cat: A14697.

[0099] HEK293F cells: purchased from Thermo-fisher, product number: A14528.

[0100] MCF-7 human breast cancer cells: purchased from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60380.

[0101] SKOV3 human ovarian cancer cells: purchased from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60291.

[0102] PC-3 human prostate cancer cells: purchased from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60343.

[0103] CT2 mouse colon cancer cells: purchased from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60043.

[0104] MC38 mouse colon cancer cells: purchased from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60825.

[0105] Example 1. Parental monoclonal antibody and positive control antibody

[0106] According to patent search, the parent monoclonal antibody Anti-B7H3, the parent monoclonal antibody Anti-4-1BB and the positive control antibody Urelumab analogue were designed for control in subsequent experiments, and the specific amino acid sequences are shown in Table 1. The specific preparation methods of each control antibody can be referred to Example 3.

[0107] Table 1. Amino acid sequences of control antibodies of the present invention

[0108]

[0109]

[0110] Example 2. Design of anti-human B7H3 / 4-1BB bispecific antibody

[0111] The anti-human B7H3 / 4-1BB bispecific antibody comprises two parent antibodies: a B7H3 antigen-binding domain and a 4-1BB antigen-binding domain.

[0112] The parent of the B7H3 antigen binding domain is the anti-human B7H3 antibody anti-B7H3, wherein the sequence is from patent application PCT / US / 2015 / 047013, and the amino acid sequence of its heavy chain variable region is SEQ ID No.1, positions 1-118, wherein positions 26-33 (GYTFTNYD), positions 51-58 (IFPGDDST), and positions 97-107 (ARQTTGTWFAY) of SEQ ID No.1 are respectively the three CDR regions of the heavy chain variable region of the anti-human B7H3 antibody anti-B7H3. The amino acid sequence of the light chain variable region is SEQ ID No.2, positions 27-32 (QSISDY), positions 50-52 (YAS), and positions 89-97 (QNGHSFPLT) of SEQ ID No.2 are respectively the three CDR regions of the light chain variable region of the anti-human B7H3 antibody anti-B7H3.

[0113] The parent of the 4-1BB antigen binding domain is the anti-human 4-1BB monoclonal antibody anti-4-1BB. The anti-human 4-1BB monoclonal antibody anti-4-1BB was independently created by Hefei Hanke Maibo Biotechnology Co., Ltd., and its sequence was disclosed in patent application WO2021093753A1. The amino acid sequence of the heavy chain variable region of the anti-human 4-1BB monoclonal antibody anti-4-1BB is positions 590-706 of SEQ ID No. 1, wherein positions 620-624 (SYGVH), positions 639-654 (VIWPGGSTNYNSALMS) and positions 687-695 (VTGTWYFDV) of SEQ ID No. 1 are respectively the three CDR regions of the heavy chain variable region; the amino acid sequence of the light chain variable region of the anti-4-1BB antibody is positions 463-569 of SEQ ID No. 1, wherein positions 486-496 (SASQGISNYLN), positions 512-518 (YTSTLHS) and positions 551-559 (QQYSKLPWT) of SEQ ID No. 1 are respectively the three CDR regions of the light chain variable region.

[0114] Design of bispecific antibody: The 4-1BB antigen binding domain is connected to the C-terminus of the anti-B7H3 antibody through a connecting peptide L1. The 4-1BB antigen binding domain is in the form of scFv, specifically, the heavy chain variable region in the 4-1BB antigen binding domain is connected to the C-terminus of the light chain variable region in the 4-1BB antigen binding domain through a connecting peptide L2 (VL-L1-VH). A pair of disulfide bonds is introduced between VL and VH in the scFv, wherein the disulfide bond position is VH44-VL100, that is, the 44th (Kabat numbering) G (glycine) of the amino acid sequence of the heavy chain variable region in the 4-1BB antigen binding domain is mutated to C (cysteine), and the 100th (Kabat numbering) G (glycine) of the amino acid sequence of the light chain variable region in the 4-1BB antigen binding domain is mutated to C (cysteine), and the amino acid sequence of the heavy chain variable region in the mutated 4-1BB antigen binding domain is positions 590-706 of SEQ ID No. 1, wherein positions 620-624 (SYGVH), 639-654 (VIWPGGSTNYNSALMS) and 687-695 (VTGTWYFDV) of SEQ ID No. 1 are three CDRs of the heavy chain variable region in the mutated 4-1BB antigen binding domain, respectively. The amino acid sequence of the light chain variable region in the mutated 4-1BB antigen binding domain is positions 463-569 of SEQ ID No. 1, wherein positions 486-496 (SASQGISNYLN), positions 512-518 (YTSTLHS) and positions 551-559 (QQYSKLPWT) of SEQ ID No. 1 are three CDRs of the light chain variable region in the mutated 4-1BB antigen binding domain, respectively.

[0115] The connecting peptides L1 and L2 are selected from the following: (G4S)n, wherein n=0, 1, 2 or 3; wherein the heavy chain constant region is any one of IgG1, IgG2, IgG3 and IgG4, or a mutant of IgG1 with H229Y or D226S and H229S mutations, or a mutant of IgG4 with S228P, F234A and L235A mutations. In addition, we also designed a mock antibody, hereinafter referred to as HK056-001 mock or mock, which is based on HK056-001, in which the antibody targeting B7H3 is partially replaced with an antibody targeting fluorescein isothiocyanate (FITC), so it does not have B7H3 targeting and is a negative control antibody of HK056-001.

[0116] The structural diagram of a bispecific antibody is shown in Figure 1 As shown in , the structural information is shown in Table 2, and the sequence is shown in Table 3:

[0117] Table 2. Structural information of bispecific antibodies

[0118]

[0119]

[0120] Table 3. Amino acid sequences of bispecific antibodies

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Example 3. Preparation of bispecific antibodies

[0127] The full-length nucleotide sequence encoding the heavy chain of the corresponding bispecific antibody was synthesized (see Table 3), and an XbaI restriction site (TCTAGA), a kozak co-recognition sequence (5'-GCCACC-3'), and a signal peptide sequence (5'-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3') were introduced at the N-terminus in sequence, and a stop codon and a HindIII restriction site (AAGCTT) were introduced at the C-terminus. The synthesized sequence was double-digested with XbaI and HindIII and then inserted into the pcDNA3.4 vector that had also been digested with enzymes. The heavy chain recombinant expression vector of the bispecific antibody was obtained by sequencing verification.

[0128] The full-length nucleotide sequence encoding the light chain of the corresponding bispecific antibody was synthesized (see Table 3), and an XbaI restriction site (TCTAGA), a kozak co-recognition sequence (5'-GCCACC-3'), and a signal peptide sequence (5'-ATGGAGACCGACACACTGCTCCTGTGGGTCCTGCTCCTCTGGGTGCCAGGAAGTACAGGA-3') were introduced at the N-terminus in sequence, and a stop codon and a HindIII restriction site (AAGCTT) were introduced at the C-terminus. The synthesized sequence was double-digested with XbaI and HindIII and then inserted into the pcDNA3.4 vector that had also been digested with enzymes. The light chain recombinant expression vector of the bispecific antibody was obtained by sequencing verification.

[0129] The light chain recombinant expression vector and the heavy chain recombinant expression vector were introduced into the human embryonic kidney cell line HEK293F at a molar ratio of 2:1, and cultured in a 37°C, 5% CO2 shaking incubator at a speed of 120rpm. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. The Protein A column (GE) was first equilibrated with PBS, and then the culture supernatant was passed through the column. First, 5 column volumes of pre-elution were performed using solution A (formula: solvent is water, solute and concentration are: 20mM sodium phosphate, 500mM NaCl, pH5.0), and then 5 column volumes of elution were performed using solution B (formula: solvent is water, solute and concentration are: 20mM sodium acetate, 150mM NaCl, pH3.5). The elution peaks were collected, and then concentrated in a 30KDa concentrating centrifuge tube to obtain the corresponding bispecific antibody molecules. The SDS-PAGE identification results are shown as follows. Figure 2 .

[0130] After this step, bispecific antibodies HK056-001 and HK056-002 were prepared for subsequent effect verification experiments.

[0131] Example 4: FACS determination of the binding properties of bispecific antibody molecules to tumor cells expressing B7H3

[0132] MCF-7, SKOV3 and PC-3 tumor cells were cultured to the logarithmic growth phase, digested with trypsin, and centrifuged at 1000 rpm for 5 min at 4°C to precipitate the cells, which were then resuspended in PBS and adjusted to 2×10 5 Each tube was washed once with 1% BSA (dissolved in PBS) solution, and the supernatant was discarded. The antibodies to be tested (bispecific antibodies HK056-001 and HK056-002, and anti-B7H3 as a positive control) were diluted to 50nM with 1% BSA as the starting concentration, and then diluted 3-fold to 8 points. 100μl of each concentration of antibody dilution was added to the EP tube containing cells, and incubated at 4℃ for 1 hour in the dark. IgG diluted with 1% BSA was used as a negative control. After incubation, 400μl of PBS containing 2% BSA was added, centrifuged for 5 minutes, and the supernatant was discarded. This operation was repeated once. Add 100 μl of 200-fold diluted goat anti-human IgG-APC (Jackson) to each EP tube, incubate at room temperature for 0.5 h, add 400 μl of 1×PBS containing 2% BSA, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat this operation once, wash, add 400 μl 1×PBS to resuspend the cells, detect on a flow cytometer, and use GraphPad Prism statistical software to process the data and calculate EC50.

[0133] The results are as follows Figure 3As shown, the bispecific antibodies HK056-001 and HK056-002 can bind to tumor cells with different B7H3 expression levels, and the binding ability is slightly lower than that of the parental monoclonal antibody anti-B7H3.

[0134] Example 5: FACS determination of the binding characteristics of bispecific antibody molecules to CHOK1 / 4-1BB cells

[0135] The method for constructing CHOK1 / 4-1BB cells is as follows: artificially synthesize the target sequence encoding the full length of human 4-1BB (Uniprot#Q07011, extracellular segment 24L-186Q) (the nucleotide sequence encoding Hu4-1BB in Table 4). Then, according to the general method in the art, the target sequence is used to replace the small fragment between the XbaⅠ and HindⅢ restriction recognition sites of the pCDNA3.4 vector to obtain the recombinant plasmid pCDNA3.4-4-1BB. The obtained recombinant plasmid pCDNA3.4-4-1BB is verified to be correct by sequencing and then introduced into wild-type CHO-K1 cells using Lipofectamine 3000 transfection reagent (Invitrogen Company) to obtain the cell line CHO-K1 / 4-1BB that highly expresses human 4-1BB.

[0136] CHOK1 / 4-1BB cells were cultured to the logarithmic growth phase, digested with trypsin, and collected in a centrifuge tube. Centrifuged at 4°C, 1000 rpm for 5 min, and PBS was added to resuspend the cells. The cells were adjusted to 2×10 5 Each tube was added with gradient dilutions of the antibodies to be tested (bispecific antibodies HK056-001 and HK056-002, and anti-4-1BB as a positive control), and the remaining steps were as described in Example 4.

[0137] The results are as follows Figure 4 As shown, both bispecific candidate molecules HK056-001 and HK056-002 can bind to CHOK1 cells expressing 4-1BB, and the binding EC50 and maximum binding values ​​are not as good as those of the parental monoclonal antibody anti-4-1BB.

[0138] Table 4. Antigenic protein related sequences

[0139]

[0140]

[0141]

[0142] Example 6: FACS determination of the binding properties of bispecific antibody molecules to B7H3 4-1BB antigens

[0143] MCF-7 tumor cells and CHOK1 / 4-1BB cells (see Example 5) were cultured to the logarithmic growth phase, digested with trypsin, and centrifuged at 1000 rpm for 5 min at 4°C to precipitate the cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 1×10 7 / ml, 1×10 7 / ml CHO-K1 / 4-1BB, and labeled with 1×10 7 / ml MCF-7. Then the antibodies to be tested (IgG group, anti-B7H3 and anti-4-1BB combination group, bispecific antibody HK056-001 group, HK056-002 group) were diluted to 150nM, and the concentration of each monoclonal antibody in the two monoclonal antibody combination groups was 150nM. Then, 100μl per tube was added to MCF-7 cells and / or CHO-K1 / 4-1BB cells and incubated at 4℃ for 1h. Finally, 100μl PBS buffer was added to each tube for detection.

[0144] The results are as follows Figure 5 As shown, both bispecific candidate molecules HK056-001 and HK056-002 can effectively mediate the simultaneous binding of MCF-7 tumor cells and CHOK1 / 4-1BB cells.

[0145] Example 7: In vitro efficacy test of the activation effect of bispecific antibodies on PBMC

[0146] A target cell-dependent PBMC activation assay was used to evaluate the activation effect of the bispecific antibody on PBMC when the target cells expressed the B7H3 antigen. The target cells used were the MCF-7 tumor cell line and the CHO-K1 Chinese hamster ovarian cancer cell line, and the effector cells used were PBMC.

[0147] Whole blood was collected from healthy subjects, and PBMCs were collected using lymphocyte separation solution (Sigma) according to the instructions. CD3 antibody OKT3 (Biolegend, catalog number 317325) was diluted to 800 ng / ml or 80 ng / ml with PBS, added to a 96-well plate, and incubated at 37°C for 2 h. The purpose of adding OKT3 was to provide the first signal to stimulate T cells. After removing the supernatant and washing with PBS, MCF-7 (highly expressing B7H3) or CHO-K1 (not expressing B7H3) and PBMC were added to a 96-well plate at a ratio of effector cells to target cells (referred to as effector-target ratio) of 3:1 or 1:3, and the target cells were fixed at 1×10 4Each well has different effector-target ratios to adjust the effector cells. Add the test antibodies (bispecific antibodies HK056-001 and HK056-002, and Urelumab antibody as a positive control) in gradient dilutions at the same time as the cells. See the dilution multiples for details. Figure 6 After incubation at 37°C for 3 days, the expression level of IFN-γ in the supernatant was detected.

[0148] The results are as follows Figure 6 As shown, the bispecific antibody induced IFN-γ secretion from PBMCs only in the presence of B7H3, compared with the negative control IgG.

[0149] Example 8: Reporter gene detection of the ADCC effect of bispecific antibodies on B7H3

[0150] Jurkat / FcγRIIIa(158V) / luc cells (Promega) were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to a 96-well plate at 3×10 cells per well. 4 MCF-7 cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to the corresponding 96-well plate, with 3×10 cells per well. 4 The samples to be tested (bispecific antibody HK056-001 group, HK056-002 group, anti-B7H3 group, HK056-001 mock group and IgG group) were diluted to 60 nM, 4-fold gradient dilution was performed at 7 points, added to a 96-well plate, incubated in a 37°C incubator for 18 h, and then 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. After incubation at room temperature for 10 min, the chemiluminescence value was detected.

[0151] The results are as follows Figure 7 As shown, the IgG4 subtype bispecific antibody HK056-002 has no ADCC effect on MCF-7 cells; HK056-001 has a significant ADCC effect in the presence of MCF-7 cells.

[0152] Example 9: Detection of the ADCC killing effect of human PBMC on tumor cells mediated by bispecific antibodies

[0153] PBMCs were isolated from fresh blood of healthy subjects using lymphocyte separation solution (Sigma) according to the instructions and incubated at 37°C overnight. The samples to be tested (bispecific antibody HK056-001 group, HK056-002 group, anti-B7H3 group, HK056-001mock group and IgG group) were diluted to 5 nM, 10-fold gradient dilutions were made into 5 points, and added to 96-well plates. MCF-7, SKOV3 and PC-3 cells were cultured to the logarithmic growth phase, digested and counted, and then labeled with Calcein AM (manufacturer YEASEN, catalog number 40719ES80). The labeled target cells were added to 96-well plates, and 1×10 4 10 cells and incubate in a 37°C incubator in the dark for 30 min. Take out the PBMC cells incubated overnight in the incubator, wash them once with 1640 medium, resuspend them and count them, and add 3×10 cells per well of a 96-well plate. 5 PBMC. After centrifugation at 100g for 2 minutes, place in a 37℃ incubator incubated in the dark for 3 hours. After centrifugation at 1000rpm for 5 minutes, take the supernatant to another 96-well plate, and detect the fluorescence value with a bioteck microplate reader. Calculate the cell mortality rate according to the following formula: Cell mortality rate (%) = (fluorescence value of the drug group-SR) / (MR-SR). Among them, MR is the fluorescence value of the maximum release well, and SR is the fluorescence value of the spontaneous release well. Both groups only have labeled target cells, and no effector cell PBMC is added. Among them, the cells in the maximum release well are added with 10% TritonX-100 30 minutes before reading the plate and continue to be cultured in a 37℃ incubator for the remaining 30 minutes. The cells in the spontaneous release well are not treated in any way, and the supernatant is directly taken after the final centrifugation to read the fluorescence value.

[0154] The results are as follows Figure 8 As shown, HK056-001 has the ability to mediate PBMC to kill tumor cells through ADCC effect, which is slightly weaker than anti-B7H3. HK056-002 has no ADCC killing effect.

[0155] Example 10: Detection of the direct killing effect of human PBMC on tumor cells mediated by bispecific antibodies

[0156] A target cell-dependent PBMC killing of tumor cells experiment was used to evaluate the effect of the bispecific antibody in activating PBMC to kill tumor cells when the target cells expressed the B7H3 antigen. The target cells used were the MCF-7 tumor cell line and the CHO-K1 Chinese hamster ovarian cancer cell line, and the effector cells used were PBMC.

[0157] Whole blood was collected from healthy individuals, and PBMCs were collected using lymphocyte separation solution (Sigma) according to the instructions. CD3 antibody (Biolegend, Cat. No. 317325) was diluted to 800 ng / ml or 80 ng / ml with PBS, added to a 96-well plate, and incubated at 37°C for 2 h. The supernatant was removed, washed with PBS, and MCF-7 or CHO-K1 and PBMC were added to a 96-well plate at a ratio of effector cells to target cells (abbreviated as effector-target ratio) of 3:1 or 1:3, where the target cells were 1×10 4 Each well has different effector-target ratios to adjust the effector cells. Add the test antibodies (bispecific antibodies HK056-001 and HK056-002, and Urelumab antibody as a positive control) in gradient dilutions at the same time as the cells. See the dilution multiples for details. Fig. 9 After incubation at 37°C for 3 days, the activity of tumor cells was detected using CCK-8 reagent (Dongren Chemical), and the killing rate of PBMC on tumor cells was calculated.

[0158] The results are as follows Fig. 9 As shown, both HK056-001 and HK056-002 have a significant killing effect on the MCF-7 tumor cell line expressing B7H3, but have no killing effect on the B7H3-negative CHO-K1 cells.

[0159] Example 11: Reporter gene detection of the activation of the 4-1BB signaling pathway by bispecific antibodies

[0160] The HEK293 / 4-1BB / NFkB-luc cell construction method is as follows: the recombinant plasmid pCDNA3.4-4-1BB prepared in Example 5 and the plasmid B (pNF-κB-Luc, Ubest Biotech, product number VT1588) having the NFκB element sequence and the luciferase gene are introduced into HEK293 cells (Shanghai Cell Bank, Chinese Academy of Sciences) using Lipofectamine 3000 transfection reagent (Invitrogen). HEK293 / 4-1BB / NFkB-luc is obtained by pressure screening.

[0161] HEK293 / 4-1BB / NFkB-luc cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to a 96-well plate at 3×10 cells per well. 4 MCF-7 cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to the corresponding 96-well plate, with 3×10 cells per well. 4The samples to be tested (bispecific antibody HK056-001 group, HK056-002 group, anti-B7H3+anti-B7H3 combination group, HK056-001 mock group and IgG group) were diluted to 60 nM (the concentrations of the two monoclonal antibodies in the anti-B7H3+anti-B7H3 combination group were both 60 nM), 4-fold gradient dilutions were performed at 7 points, added to a 96-well plate, incubated in a 37°C incubator for 18 h, and then 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. The chemiluminescence value was detected after incubation at room temperature for 10 min.

[0162] The results are as follows Fig.10 As shown, both HK056-001 and HK056-002 can effectively activate the 4-1BB signaling pathway, while the combination of the two parental monoclonal antibodies alone cannot activate the downstream 4-1BB signaling pathway.

[0163] Example 12, in vivo efficacy test of the inhibitory effect of bispecific antibodies on CT26 / HuB7H3 tumor growth Construction of CT26 / HuB7H3: artificially synthesized the target sequence encoding the full length of human B7H3 (Uniprot#Q5ZPR3) (the nucleotide sequence encoding HuB7H3 in Table 4). Then, according to the general method in the art, the target sequence was used to replace the small fragment between the XbaⅠ and HindⅢ restriction recognition sites of the pCDNA3.4 vector to obtain the recombinant plasmid pCDNA3.4-B7H3. The obtained recombinant plasmid pCDNA3.4-B7H3 was verified to be correct by sequencing and then introduced into wild-type CT26 cells using Lipofectamine3000 transfection reagent (Invitrogen) to obtain the cell line CT26 / HuB7H3 that highly expresses human B7H3.

[0164] CT26 / HuB7H3 cells were cultured in vitro and digested with trypsin. 6 6-8 week old Balb / c-h4-1BB mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Strain No. T003359) were subcutaneously inoculated and grouped and dosed 6 days after inoculation (average tumor volume was about 72 mm 3 ), 5 mice per group, intraperitoneal administration (100 μl per mouse), twice a week, for a total of 6 times. The administration groups and dosages are as follows:

[0165] 1) Normal saline;

[0166] 2) HK056-001, 2 mg / kg;

[0167] 3) HK056-001, 8 mg / kg;

[0168] 4) HK056-002, 8 mg / kg;

[0169] 5)anti-B7H3+anti-4-1BB, 6+6mg / kg;

[0170] 6) HK056-001+mPD-1,2+5mg / kg;

[0171] 7) mPD-1,5mg / kg.

[0172] Among them, mPD-1 is an anti-mouse PD-1 antibody (manufacturer: Bio X cell, catalog number: BE0146-100MG).

[0173] The drug was given twice a week and the animal survival and activity were checked and recorded, including tumor growth, body weight, activity, and diet. The tumor volume was calculated using the formula 1 / 2×length×width×width (mm 3 ).

[0174] The experimental results are as follows Fig.11 As shown, HK056-001 had a good tumor inhibitory effect at both 2 mg / kg and 8 mg / kg, with complete tumor disappearance (CR) in 1 and 2 mice, respectively. The efficacy was better than the 8 mg / kg dose group of HK056-002 and the combination group of two parental monoclonal antibodies; and it showed a better anti-tumor effect when used in combination with PD-1 antibodies, with complete tumor disappearance in 4 mice.

[0175] Example 13, Challenge experiment of CT26 / HuB7H3 tumor model

[0176] The cured (CR) mice in Example 12 were observed for at least one month, and if the tumor did not recur, a challenge experiment was performed: CT26 wild-type cells were cultured in vitro, digested with trypsin, and then incubated at 1.25×10 6 / Balb / c-h4-1BB mice that were cured (CR) after subcutaneous inoculation were observed for 39 days, and no tumor growth was observed, indicating that the treated mice had immune memory and the tumor would not recur after challenge. Fig.12 shown.

[0177] Example 14: In vivo efficacy test of the inhibitory effect of bispecific antibodies on MC38 / HuB7H3 tumor growth

[0178] Construction of MC38 / HuB7H3 cell line: pCDNA3.4-B7H3 plasmid was synthesized according to the method in Example 12 and introduced into wild-type MC38 cells using Lipofectamine 3000 transfection reagent (Invitrogen) to obtain the cell line MC38 / HuB7H3 that highly expresses human B7H3.

[0179] MC38 / HuB7H3 cells were cultured in vitro and digested with trypsin. 6 8-9 week old B6-hCD137 mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Strain No. T003360) were subcutaneously inoculated and grouped and dosed 7 days after inoculation (average tumor volume was about 52 mm 3 ), 6 mice per group, intraperitoneal administration (100 μl per mouse), twice a week, 5 times in total. The administration groups and dosages are as follows:

[0180] 1) Normal saline;

[0181] 2) HK056-001, 3 mg / kg;

[0182] 3) HK056-001, 1 mg / kg;

[0183] 4) HK056-001, 0.3 mg / kg;

[0184] The experimental results are as follows Fig.13 As shown, HK056-001 exhibited significant dose-dependent antitumor activity in 4-1BB humanized mice inoculated with MC38-HuB7H3.

[0185] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A bispecific antibody targeting B7H3 and 4-1BB, formed by connecting a 4-1BB antigen binding domain to the C-terminus of each of the two heavy chains of an anti-B7H3 antibody; The 4-1BB antigen binding domain is an anti-4-1BB single-chain antibody, including the heavy chain variable region of the anti-4-1BB antibody and the light chain variable region of the anti-4-1BB antibody.

2. The bispecific antibody according to claim 1, characterized in that: The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-B7H3 antibody are shown in SEQ ID No. 1, 47-66, and 99-107, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-B7H3 antibody are shown in SEQ ID No. 2, 50-56, and 89-97, respectively; and / or The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-4-1BB antibody are shown in positions 620-624 of SEQ ID No.1, positions 639-654 of SEQ ID No.1, and positions 687-695 of SEQ ID No.1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-4-1BB antibody are shown in positions 486-496 of SEQ ID No.1, positions 512-518 of SEQ ID No.1, and positions 551-559 of SEQ ID No.1, respectively.

3. The bispecific antibody according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-B7H3 antibody is shown in positions 1-118 of SEQ ID No.1; the amino acid sequence of the light chain variable region of the anti-B7H3 antibody is shown in positions 1-107 of SEQ ID No.

2.

4. The bispecific antibody according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-4-1BB antibody is shown in positions 590-706 of SEQ ID No. 1; the amino acid sequence of the light chain variable region of the anti-4-1BB antibody is shown in positions 463-569 of SEQ ID No.

1.

5. The bispecific antibody according to claim 4, characterized in that: The amino acid sequence of the 4-1BB antigen binding domain is shown in positions 463-706 of SEQ ID No.

1.

6. The bispecific antibody according to claim 5, characterized in that: The amino acid sequence of one chain of the bispecific antibody is shown in SEQ ID No.1, and the amino acid sequence of the other chain is shown in SEQ ID No.

2.

7. Any of the following substances: (A1) a nucleic acid molecule encoding the bispecific antibody according to any one of claims 1 to 6; (A2) an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule described in (A1); (A3) A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 6.

8. The substance according to claim 7, characterized in that: In (A1), the nucleotide sequence encoding the heavy chain variable region of the anti-4-1BB antibody is shown in positions 1768-2118 of SEQ ID No. 3; the nucleotide sequence encoding the light chain variable region of the anti-4-1BB antibody is shown in positions 1387-1707 of SEQ ID No. 3; and / or The nucleotide sequence encoding the heavy chain variable region of the anti-B7H3 antibody is shown in positions 1-354 of SEQ ID No. 3; the nucleotide sequence encoding the light chain variable region of the anti-B7H3 antibody is shown in positions 1-321 of SEQ ID No. 4; Further, the nucleotide sequence encoding the 4-1BB antigen binding domain is shown in 1387-2118 of SEQ ID No.3; and / or Furthermore, the nucleotide sequence encoding one chain of the bispecific antibody is shown in SEQ ID No.3, and the nucleotide sequence encoding the other chain of the bispecific antibody is shown in SEQ ID No.

4.

9. A method for preparing the bispecific antibody according to any one of claims 1 to 6, comprising the following steps: introducing the nucleic acid molecule encoding the bispecific antibody according to claim 7 or 8 into a host cell, and obtaining the bispecific antibody after culturing.

10. Use of the bispecific antibody or substance according to any one of claims 1 to 8 in any of the following: (B1) Preparation of antibody targeted drugs; (B2) preparing a product for activating T cells; (B3) preparing products that kill tumor cells; (B4) Preparation of products for preventing and / or treating tumors.

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