CN119462951A - A bispecific antibody targeting B7H3 and 4-1BB and its application - Google Patents

Abstract

The invention discloses a bispecific antibody targeting B7H3 and 4-1BB and application thereof. The invention provides a bispecific antibody targeting B7H3 and 4-1BB, which is formed by respectively connecting a 4-1BB antigen binding domain to the C ends of two heavy chains of an anti-B7H 3 antibody, wherein the 4-1BB antigen binding domain is a single-chain antibody resisting 4-1BB, and comprises a heavy chain variable region resisting 4-1BB antibody and a light chain variable region resisting 4-1BB antibody. The bispecific antibody provided by the invention has good in-vitro tumor cell killing effect and in-vivo tumor inhibiting effect, so that the bispecific antibody provided by the invention has important significance and application potential in preparing antibody targeted drugs.

Description

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

Technical Field

The invention relates to the technical field of tumor treatment and biological medicine, in particular to a bispecific antibody targeting B7H3 and 4-1BB and application thereof.

Background

Bispecific antibodies (BsAb) are also called bifunctional antibodies, and can simultaneously recognize and bind two different antigens or epitopes compared with the common antibodies which can only bind to a single target, can play a certain special biological function, even is better than the synergistic effect of the combination of the two monoclonal antibodies, so that the bispecific antibodies are increasingly one of the new hot spots for antibody drug research.

B7-H3 (CD 276/B7 RP-2), a member of the B7 ligand family (PD-L1 also belongs to this family), is overexpressed in most cancer types, but is expressed at low levels in normal tissues. B7-H3 inhibits tumor antigen-specific immune responses in malignant tissues to promote tumor growth. B7-H3 also has non-immunogenic functions such as promoting migration and invasion, angiogenesis, chemotherapy resistance, endothelial cell transformation into mesenchymal cells and affecting tumor cell metabolism. The expression of B7-H3 in tumors is related to poor prognosis, and more people are focusing on the application value of the target in tumor immunotherapy.

Costimulatory molecule 4-1BB belongs to an important member of the tumor necrosis factor receptor superfamily, and is a costimulatory signal that mediates T cell activation. The 4-1BB mediated co-stimulatory signal can enhance T cell function, and enhance T cell monitoring of tumor cells and immune defense of viral infection. Modulation of lymphocyte immune function by intervention of the 4-1BB pathway is likely to be a new immunotherapeutic pathway.

Disclosure of Invention

The invention aims to provide a bispecific antibody targeting B7H3 and 4-1BB and application thereof. The bispecific antibody can simultaneously recognize two antigen targets of CD137, namely 4-1BB and B7H 3.

The invention aims to solve the technical problem of avoiding toxic and side effects caused by large-scale activation of an immune system in a targeted treatment process.

To solve the above technical problem, in a first aspect, the present invention claims bispecific antibodies targeting B7H3 and 4-1 BB. Such bispecific antibodies include an immune agonist moiety, such as a 4-1BB antigen binding domain, that can specifically bind to a 4-1BB antigen, and a tumor targeting moiety, such as a B7H3 antigen binding domain, that can specifically bind to a tumor-associated antigen.

The bispecific antibody targeting B7H3 and 4-1BB is specifically formed by connecting the C-terminal of two heavy chains of an anti-B7H 3 antibody with a 4-1BB antigen binding domain.

Wherein the 4-1BB antigen binding domain may be linked to the C-terminus of the heavy chain of said anti-B7H 3 antibody via a linker peptide. The linker peptide may be composed of a positive integer multiple of GGGGS subunits, e.g. 1,2, 3,4, 5 or 6, preferably in the bispecific antibody the linker peptide is composed of 3 GGGGS subunits. Of course, the linking peptides described herein may also be other flexible peptides, such as A (EAAAK) ₄ ALE, KVDKKVEPKSCDKTHT, and the like.

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

Further, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-B7H 3 antibody are sequentially shown as 31-35 th site of SEQ ID No.1, 47-66 th site of SEQ ID No.1 and 99-107 th site of SEQ ID No.1, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-B7H 3 antibody are sequentially shown as 24-34 th site of SEQ ID No.2, 50-56 th site of SEQ ID No.2 and 89-97 th site of SEQ ID No. 2.

Further, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-4-1 BB antibody are sequentially shown as 620-624 th site of SEQ ID No.1, 639-654 th site of SEQ ID No.1 and 687-695 th site of SEQ ID No.1, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the anti-4-1 BB antibody are sequentially shown as 486-496 site of SEQ ID No.1, 512-518 th site of SEQ ID No.1 and 551-559 th site of SEQ ID No. 1.

In the present invention, the CDRs are "complementarity determining regions" which are regions of the antibody variable domain which are hypervariable in sequence and form structurally defined "hypervariable loops" and/or "antigen contact points" containing antigen contact residues. CDRs are mainly responsible for binding to the epitope. One variable region typically comprises 3 CDR regions, CDR1, CDR2 and CDR3 in order from the N-terminus. HCDR1, HCDR2 and HCDR3 represent 3 CDR regions in the heavy chain variable region, LCDR1, LCDR2 and LCDR3 represent 3 CDR regions in the light chain variable region.

Furthermore, the amino acid sequence of the heavy chain variable region of the anti-B7H 3 antibody is shown in positions 1-118 of SEQ ID No.1, and the amino acid sequence of the light chain variable region of the anti-B7H 3 antibody is shown in positions 1-107 of SEQ ID No. 2.

Furthermore, the amino acid sequence of the heavy chain variable region of the anti-4-1 BB antibody is shown in 590-706 of SEQ ID No.1, and the amino acid sequence of the light chain variable region of the anti-4-1 BB antibody is shown in 463-569 of SEQ ID No. 1.

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

Further, the heavy chain constant region in the bispecific antibody may be selected from any one of the following:

1) IgG, igM, igE, igA or IgD;

2) IgG1, igG2, igG3 or IgG4;

3) IgG4 mutant with S228P, F A and L235A mutations.

In one embodiment of the invention, the heavy chain constant region in the bispecific antibody is IgG1 (corresponding to antibody HK 056-001), and in another embodiment of the invention, the heavy chain constant region in the bispecific antibody is an IgG4 mutant (corresponding to antibody HK 056-002) having been subjected to S228P, F234A and L235A mutations.

Further, the bispecific antibody consists 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.

In a specific embodiment of the invention, the anti-B7H 3 antibody is from patent PCT/US/2015/047013 and the anti-4-1 BB antibody is from patent WO2021093753A1.

In a second aspect, the invention claims a substance associated with the bispecific antibody described in the first aspect above.

The substance claimed in the present invention in relation to the bispecific antibody described in the first aspect above may be any of the following:

(A1) A nucleic acid molecule encoding a bispecific antibody as described in the first aspect hereinbefore;

(A2) An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of (A1);

(A3) A pharmaceutical composition comprising a bispecific antibody as described in the first aspect hereinbefore.

Further, in (A1), the nucleotide sequence encoding the heavy chain variable region of the anti-4-1 BB antibody is shown at positions 1768-2118 of SEQ ID No.3, and the nucleotide sequence encoding the light chain variable region of the anti-4-1 BB antibody is shown at positions 1387-1707 of SEQ ID No. 3. The nucleotide sequence of the heavy chain variable region of the anti-B7H 3 antibody is shown in positions 1-354 of SEQ ID No.3, and the nucleotide sequence of the light chain variable region of the anti-B7H 3 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 as SEQ ID No.3, no. 1387-2118.

More specifically, the nucleotide sequence encoding one strand of the bispecific antibody is shown as SEQ ID No.3, and the nucleotide sequence encoding the other strand of the bispecific antibody is shown as SEQ ID No. 4.

In a specific embodiment of the invention, the recombinant vector is recombinant plasmid a and/or recombinant plasmid B. The recombinant plasmid A is obtained by cloning the coding gene (SEQ ID No. 3) of one chain of the bispecific antibody into a pcDNA3.4 vector (such as between XbaI and HindIII sites). The recombinant plasmid B is obtained by cloning the coding gene (SEQ ID No. 4) of the other chain of the bispecific antibody into a pcDNA3.4 vector (such as between 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 a human embryonic kidney cell line HEK 293F.

Further, in (A3), 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 polypeptides and chemokines, etc., may also be included in the pharmaceutical composition. In a specific embodiment of the invention, the pharmaceutical composition consists of the bispecific antibody and an anti-PD-1 antibody.

Further, in (A3), a pharmaceutically acceptable excipient, diluent or carrier may be further included in the pharmaceutical composition.

In a third aspect, the invention claims a method of preparing a bispecific antibody as described in the first aspect above.

The method of preparing the bispecific antibody according to the first aspect of the present invention may comprise the step of introducing the nucleic acid molecule encoding the bispecific antibody according to the second aspect of the present invention into a recipient cell, and culturing the cell to obtain the bispecific antibody.

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

In the method, the method further comprises the step of separating and purifying the bispecific antibody by an affinity chromatography method, wherein the bispecific antibody can be purified into a substantially homogeneous substance.

Further, the nucleic acid molecule encoding the bispecific antibody may be introduced into the recipient cell in the form of a recombinant vector. Wherein the recombinant vector may be the recombinant vector described in the second aspect above.

Further, the recipient cell may be a mammalian cell or an insect cell. The mammalian cell is, in particular, the human kidney epithelial cell line HEK293F.

In a fourth aspect, the invention claims the use of a bispecific antibody as described in the first aspect hereinbefore or a substance as described in the second aspect hereinbefore in any of the following:

(B1) Preparing an antibody targeting drug;

(B2) Preparing a T cell activating product;

(B3) Preparing a product for killing tumor cells;

(B4) Preparing the product for preventing and/or treating tumor.

Further, the product may be an antibody drug, CAR-T, CAR-NK, an antibody-conjugated drug, an immunotoxin, a vaccine, and the like.

Further, the activated T cells are activated T cells in the presence of tumor cells expressing B7H 3.

Further, the killer tumor cells are the bispecific antibody-mediated PBMC killer tumor cells. Specifically, the method can comprise the steps of mediating NK cells to kill tumor cells through ADCC effect and mediating T cells to directly kill the tumor cells.

Wherein the tumor cell is a tumor cell expressing B7H3, and the tumor is a tumor expressing B7H 3.

Further, the tumor may be selected from mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, bile duct cancer, endometrial cancer, thymus cancer, colon cancer, and breast cancer.

In a specific embodiment of the invention, the tumor cells are specifically selected from the group consisting of breast cancer cells (e.g., MCF-7 cells), ovarian cancer cells (e.g., SKOV3 cells), prostate cancer cells (e.g., PC-3 cells), colon cancer cells (e.g., B7H3 transfected CT26 cells or MC38 cells).

In a specific embodiment of the invention, the tumor is specifically selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, colorectal cancer.

The beneficial technical effects obtained by the invention are as follows:

1. the bispecific antibody targeting B7H3 and 4-1BB can simultaneously recognize two antigen targets of 4-1BB and B7H 3.

The bispecific antibody is capable of simultaneously binding B7H3 and 4-1BB.

The bispecific antibodies are capable of activating PBMCs, in particular T cells in PBMCs, and enhancing IFN- γ secretion levels in vitro experiments.

The activation effect of the bispecific antibody on PBMC is B7H3 dependent, without B7H3 activity.

The bispecific antibody is capable of activating the 4-1BB signaling pathway, which activation is dependent on cross-linking (Crosslinking).

The bispecific antibodies are capable of enhancing killing of tumor cells by NK cells and PBMCs in vitro experiments.

2. The bispecific antibody has anti-tumor efficacy, and the efficacy of the bispecific antibody is superior to that of the corresponding bispecific antibody without Fc function. The bispecific antibody has the capability of simultaneously recognizing a tumor cell surface antigen B7H3 and an immune cell surface antigen 4-1BB, and can activate immune cells through efficient enrichment of B7H3 near tumor cells, so that systemic toxicity caused by activation of systemic immune cells by 4-1BB agonist antibodies can be effectively avoided, adaptive immunity can be caused by combination with Fc receptors, and efficient anti-tumor effect can be achieved.

3. Experiments prove that the bispecific antibody provided by the invention has good T cell activating effect, and on the basis, good in-vitro tumor cell killing effect and in-vivo tumor inhibiting effect are shown, so that the bispecific antibody provided by the invention has important significance and application potential in preparing antibody targeted drugs.

Drawings

FIG. 1 is a schematic structural diagram of a bispecific antibody molecule.

FIG. 2 shows the results of electrophoresis (SDS-PAGE) of bispecific antibodies under non-reducing and reducing conditions.

FIG. 3 is a flow chart of the activity of a bispecific antibody molecule in binding to the B7H3 antigen.

FIG. 4 is a flow chart of the activity of a bispecific antibody molecule in binding to 4-1BB antigen.

FIG. 5 is a flow chart of the activity of a bispecific antibody molecule in simultaneous binding to human B7H3 antigen and human 4-1BB antigen.

FIG. 6 shows B7H3 mediated activation of PBMC by bispecific antibody molecules to produce IFN-gamma.

Figure 7 is a reporter gene to detect ADCC effect of bispecific antibody molecules on tumor cells.

Figure 8 is bispecific antibody molecule mediated ADCC killing of tumor cells by human PBMCs.

FIG. 9 is a direct killing of tumor cells by human PBMC mediated by bispecific antibody molecules.

FIG. 10 is a reporter gene detection of B7H3 mediated activation of the 4-1BB signaling pathway by a bispecific antibody molecule.

FIG. 11 shows the in vivo efficacy of bispecific antibody molecules for the inhibition of tumor growth in CT26/HuB7H3 model.

FIG. 12 is a challenge experiment of CT26/HuB7H3 tumor model.

FIG. 13 is a graph showing the in vivo efficacy of bispecific antibodies for the inhibition of MC38/HuB7H3 tumor growth.

Detailed Description

Terms and definitions

BsAb, bispecific antibody (bispecific antibody);

TAA, tumor-associated antigen (Tumor associated antibody);

VH heavy chain variable region (HEAVY CHAIN variabledomain);

VL light chain variable region (LIGHT CHAIN variabledomain);

FACS, fluorescence activated cell sorting, also known as flow cytometry (Fluorescence-ACTIVATED CELL sort);

SDS-PAGE, polyacrylamide gel electrophoresis;

PBMC, peripheral blood mononuclear cells;

CR is an abbreviation for Complete release, meaning that the tumor completely disappears.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

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

As used herein, the term EC50 refers to the half maximal effect concentration (concentration for 50%of maximal effect), which refers to the concentration that causes 50% of the maximal effect.

As used herein, the term "monoclonal Antibody" or "anti-body", unless otherwise specifically indicated, generally refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain; heavy chains are understood to be, in a general sense, polypeptide chains of greater molecular weight in an Antibody, light chains refer to polypeptide chains of lesser molecular weight in an Antibody; light chains are classifiable as kappa and lambda light chains; heavy chains are generally classifiable as mu, delta, gamma, alpha or epsilon, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively, within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, the heavy chain also comprises a "D" region of about 3 or more amino acids, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH), each heavy chain constant region is composed of 3 domains (CH 1, CH2 and CH 3), each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL), each light chain constant region is composed of one domain CL, the constant region of an Antibody can mediate immunoglobulins with host tissues or factors, the VH and VL regions can also be subdivided into regions of high variability (termed Complementarity Determining Regions (CDRs)) interspersed with regions that are more conserved, termed Framework Regions (FR), each VH and VL consisting of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consists of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows the definition of Kabat Sequences of Proteins of ImmunologicalInterest(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 may also comprise more than 3 CDRs, for example 6, 9, or 12. For example, in a bifunctional antibody of the invention, the heavy chain may be an scFv of an IgG antibody with the C-terminal end of the heavy chain linked to another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

Antigen binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened in the same manner as for intact antibodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the term "isolated" or "isolated" refers to obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" or "separated" does not exclude the presence of substances mixed with artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substances.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby 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 (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

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

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

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

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

The pcDNA3.4 vector is Invitrogen, cat: A14697.

HEK293F cells from Thermo-fisher, product No. A14528.

MCF-7 human breast cancer cells available from Nanjac, bai Biotechnology Co., ltd, product number CBP60380.

SKOV3 human ovarian cancer cells available from Nanjac, bai Biotechnology Co., ltd., product number CBP60291.

PC-3 human prostate cancer cells were purchased from Nanjac, bai Biotechnology Co., ltd, product number CBP60343.

CT2 mouse colon cancer cells were purchased from Nanjac Bai Biotechnology Co., ltd, product number CBP60043.

MC38 mouse colon cancer cells were purchased from Nanjac Bai Biotechnology Co., ltd., product number CBP60825.

Example 1, maternal monoclonal antibody and Positive control antibody

According to the patent query, the maternal monoclonal antibody-B7H 3 maternal monoclonal antibody-4-1 BB and the positive control antibody Urelumab analogue are designed for the control in the subsequent experiments, and the specific amino acid sequences are shown in Table 1. For specific preparation of each control antibody, reference is made to example 3.

TABLE 1 amino acid sequences of antibodies for control of the present invention

Example 2 design of anti-human B7H3/4-1BB bispecific antibody

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

The parent of the B7H3 antigen binding domain is anti-human B7H3 antibody anti-B7H3, wherein the sequence is from patent application PCT/US/2015/047013, the amino acid sequence of the heavy chain variable region is 1 st-118 th site of SEQ ID No.1, wherein the 26 th-33 th site (GYTFTNYD), the 51 th-58 th site (IFPGDDST) of SEQ ID No.1, and the 97 th-107 th site (ARQTTGTWFAY) are respectively 3 CDR regions of the heavy chain variable region of the anti-human B7H3 antibody anti-B7H 3. The amino acid sequence of the light chain variable region is 1 st to 107 th of SEQ ID No.2, 27 th to 32 nd (QSISDY) of SEQ ID No.2, 50 th to 52 th (YAS) and 89 th to 97 th (QNGHSFPLT) are respectively 3 CDR regions of the light chain variable region of anti-human B7H3 antibody anti-B7H3

The parent of the 4-1BB antigen binding domain is anti-human 4-1BB monoclonal antibody anti-4-1BB, and the anti-human 4-1BB monoclonal antibody anti-4-1BB is self-created by the company of the Biotechnology of Margaritifera, hefei Kagaku, the sequence of which is disclosed in patent application WO2021093753A 1. The amino acid sequence of the heavy chain variable region of the anti-human 4-1BB monoclonal antibody anti-4-1BB is 590 to 706 of SEQ ID No.1, wherein 620 to 624 (SYGVH), 639 to 654 (VIWPGGSTNYNSALMS) and 687 to 695 (VTGTWYFDV) of SEQ ID No.1 are 3 CDR regions of the heavy chain variable region respectively, and the amino acid sequence of the light chain variable region of the anti-4-1BB antibody is 463 to 569 of SEQ ID No.1, wherein 486 to 496 (SASQGISNYLN), 512 to 518 (YTSTLHS) and 551 to 559 (QQYSKLPWT) of SEQ ID No.1 are 3 lattice CDR regions of the light chain variable region respectively.

Bispecific antibody design the 4-1BB antigen binding domain is linked to the C-terminus of the anti-B7H 3 antibody via a linker peptide L1. Wherein the 4-1BB antigen-binding domain is in the form of a scFv, in particular, the heavy chain variable region in the 4-1BB antigen-binding domain is linked to the C-terminus of the light chain variable region in the 4-1BB antigen-binding domain (VL-L1-VH) by a linking peptide L2. A pair of disulfide bonds is introduced between VL and VH in the scFv, wherein the disulfide bond position is VH44-VL100, namely G (glycine) at position 44 (Kabat numbering) of the amino acid sequence of the heavy chain variable region in the 4-1BB antigen binding domain is mutated to C (cysteine), G (glycine) at position 100 (Kabat numbering) of the amino acid sequence of the light chain variable region in the 4-1BB antigen binding domain is mutated to C (cysteine), the amino acid sequence of the heavy chain variable region in the mutated 4-1BB antigen binding domain is 590-706 of SEQ ID No.1, wherein the amino acid sequence of SEQ ID No.1 is the three CDRs of the heavy chain variable region in the mutated 4-1BB antigen binding domain at positions 620-624 (SYGVH), 639-654 (VIWPGGSTNYNSALMS) and 687-695 (VTGTWYFDV), 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), 512-518 (YTSTLHS) and 551-559 (QQYSKLPWT) of SEQ ID No.1 are the three CDRs of the light chain variable region in the mutated 4-1BB antigen binding domain, respectively.

The connecting peptide L1 and L2 is selected from (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 subjected to H229Y or D226S and H229S mutation, or a mutant of IgG4 subjected to S228P, F234A and L235A mutation. In addition, we also designed a mock antibody, hereinafter referred to as HK056-001 mock or mock, which was a negative control antibody of HK056-001, in which the B7H 3-targeting antibody moiety was replaced with a Fluorescein Isothiocyanate (FITC) -targeting antibody based on HK056-001, and thus did not have B7H3 targeting.

The structural schematic of the bispecific antibody is shown in fig. 1, the structural information is shown in table 2, and the sequence is shown in table 3:

TABLE 2 structural information of bispecific antibodies

TABLE 3 amino acid sequences of bispecific antibodies

Example 3 preparation of bispecific antibodies

Synthesizing a nucleotide full-length sequence (see table 3) for encoding a heavy chain of the corresponding bispecific antibody, sequentially introducing an XbaI enzyme cutting site (TCTAGA), a kozak consensus sequence (5 '-GCCACC-3') and a signal peptide sequence (5'-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3') at the N end, introducing a stop codon and a HindIII enzyme cutting site (AAGCTT) at the C end, and inoculating the synthesized sequence to a pcDNA3.4 vector which is subjected to enzyme cutting after double enzyme cutting by the XbaI and HindIII, and obtaining the heavy chain recombinant expression vector of the bispecific antibody through sequencing verification.

Synthesizing a nucleotide full-length sequence (see table 3) for encoding a light chain of a corresponding bispecific antibody, sequentially introducing an XbaI enzyme cutting site (TCTAGA), a kozak consensus sequence (5 '-GCCACC-3') and a signal peptide sequence (5'-ATGGAGACCGACACACTGCTCCTGTGGGTCCTGCTCCTCTGGGTGCCAGGAAGTACAGGA-3') at the N end, introducing a stop codon and a HindIII enzyme cutting site (AAGCTT) at the C end, and inoculating the synthesized sequence to a pcDNA3.4 vector which is subjected to enzyme cutting after double enzyme cutting by the XbaI and HindIII, and obtaining the light chain recombinant expression vector of the bispecific antibody through sequencing verification.

The light chain recombinant expression vector and the heavy chain recombinant expression vector were introduced into a human embryonic kidney cell line HEK293F at a molar ratio of 2:1, and cultured in a shaking incubator at 37℃and 5% CO ₂ at a speed of 120rpm. Antibody proteins were purified from the culture supernatant using Protein a affinity chromatography column. The Protein A column (GE company) is equilibrated by PBS, the supernatant is cultured and passed through the column, 5 column volumes are pre-eluted by adopting A solution (formula: solvent is water, solute and concentration are 20mM sodium phosphate, 500mM NaCl, pH 5.0), 5 column volumes are eluted by adopting B solution (formula: solvent is water, solute and concentration are 20mM sodium acetate, 150mM NaCl, pH 3.5), eluting peaks are collected, and then 30kDa is concentrated by a centrifuge tube, and the corresponding bispecific antibody molecule is obtained. SDS-PAGE was identified as shown in FIG. 2.

Bispecific antibodies HK056-001 and HK056-002 were prepared through this step for subsequent efficacy validation experiments.

EXAMPLE 4 FACS determination of the binding Properties of bispecific antibody molecules to B7H 3-expressing tumor cells

MCF-7, SKOV3 and PC-3 tumor cells were cultured to the logarithmic growth phase, after pancreatin digestion, the cells were pelleted by centrifugation at 1000rpm for 5min at 4℃and resuspended in PBS, the cells were adjusted to 2X 10 ⁵ cells/tube, washed once with 1% BSA (in PBS) solution, and the supernatant was removed. The antibodies to be tested (bispecific antibodies HK056-001 and HK056-002, and anti-B7H3 as positive control) were diluted with 1% BSA to 50nM as starting concentration, diluted 3-fold in 8 spots, 100. Mu.l of each concentration of antibody dilution was added to the EP tube containing the cells, incubated at 4℃for 1 hour in the absence of light, and 1% BSA diluted IgG was used as negative control. After incubation 400 μl of 2% BSA in PBS was added and centrifuged for 5min and the supernatant was discarded and the procedure repeated. 100. Mu.l of goat anti-human IgG-APC (Jackson Co.) diluted 200-fold per EP tube was incubated for 0.5 hours at room temperature, 400. Mu.l of 1 XPBS with 2% BSA was added, centrifuged at 2000rpm for 5 minutes to discard the supernatant, the procedure was repeated once, after washing, 400. Mu.l of 1 XPBS was added to resuspend cells, the detection was performed by an upflow cytometer, the data was processed with GRAPHPAD PRISM statistical software and EC50 was calculated.

As shown in FIG. 3, bispecific antibodies HK056-001 and HK056-002 both bind to tumor cells with different expression levels of B7H3 with slightly lower binding capacity than the parent monoclonal antibody anti-B7H3.

EXAMPLE 5 FACS determination of binding Properties of bispecific antibody molecules to CHOK1/4-1BB cells

The CHOK1/4-1BB cell was constructed by artificially synthesizing the sequence of interest (the nucleotide sequence encoding Hu4-1BB in Table 4) encoding the full length of human 4-1BB (uniprot#Q07011, extracellular domain 24L-186Q). Then, the small fragment between the XbaI and HindIII cleavage recognition sites of the pCDNA3.4 vector was replaced with the target sequence according to the general method in the art to obtain recombinant plasmid pCDNA3.4-4-1BB, and the obtained recombinant plasmid pCDNA3.4-4-1BB was introduced into wild-type CHO-K1 cells with Lipofectamine 3000 transfection reagent (Invitrogen) after the correctness of the sequencing verification to obtain cell strain CHO-K1/4-1BB highly expressing human 4-1BB.

CHOK1/4-1BB cells were cultured to logarithmic growth phase, digested with pancreatin and collected in centrifuge tubes. Cells were resuspended at 4℃at 1000rpm for 5min and PBS was added, the cells were adjusted to 2X 10 ⁵ cells/tube, the gradient diluted antibodies to be tested (bispecific antibodies HK056-001 and HK056-002, and anti-4-1BB as positive control) were added, and the remaining steps were described in example 4.

As a result, as shown in FIG. 4, both bispecific candidate molecules HK056-001 and HK056-002 were able to bind to CHOK1 cells expressing 4-1BB with less than the parent monoclonal antibody anti-4-1BB in terms of the EC50 and maximum binding value.

TABLE 4 antigen protein related sequences

EXAMPLE 6 FACS determination of bispecific antibody molecules while binding Properties to B7H3 4-1BB antigen

MCF-7 tumor cells and CHOK1/4-1BB cells (see example 5) were cultured to logarithmic growth phase, and after pancreatin digestion, the cells were pelleted by centrifugation at 1000rpm for 5min at 4℃and adjusted to a cell concentration of 1X 10 ⁷/ml in PBS buffer, labeled 1X 10 ⁷/ml CHO-K1/4-1BB at a final concentration of CFSE (Aibisin (Shanghai) Biotech Co.) at 4. Mu.M, and labeled 1X 10 ⁷/ml MCF-7 at a final concentration of 4. Mu. M CELLTRACKER DEEP RED (Invitrogen). The antibodies to be tested (IgG group, anti-B7H3 and anti-4-1BB combination group, bispecific antibody HK056-001 group, HK056-002 group) were then diluted to 150nM, wherein the concentration of each of the two monoclonal antibodies in the combination group was 150nM, and then added to MCF-7 cells and/or CHO-K1/4-1BB cells at 100 μl per tube, and incubated at 4℃for 1H. Finally, each tube was supplemented with 100. Mu.l PBS buffer for on-line detection.

As shown in FIG. 5, both bispecific candidate molecules HK056-001 and HK056-002 were effective in mediating simultaneous binding of MCF-7 tumor cells and CHOK1/4-1BB cells.

EXAMPLE 7 in vitro efficacy detection of the activation Effect of bispecific antibodies on PBMC

Target cell-dependent PBMC activation experiments were used to evaluate the activation effect of bispecific antibodies on PBMC in the case of target cell expression of B7H3 antigen, using MCF-7 tumor cell lines and CHO-K1 chinese hamster ovary cancer cell lines, using PBMC as effector cells.

Whole blood was collected from healthy persons and PBMCs were collected using lymphocyte isolates (Sigma) according to the instructions. The CD3 antibody OKT3 (Biolegend, cat. 317325,) was diluted to 800ng/ml or 80ng/ml with PBS, added to 96-well plates, incubated at 37℃for 2h, and OKT3 was added to provide the first signal to stimulate T cells. After removing the supernatant and washing with PBS, MCF-7 (high expression B7H 3) or CHO-K1 (non-expression B7H 3) and PBMC are added into 96-well plates according to the ratio of effector cells to target cells (short effective target ratio) of 3:1 or 1:3, wherein the target cells are fixed into 1X 10 ⁴ target cells/well, and different effective target ratios regulate the effector cells. The test antibodies (bispecific antibodies HK056-001 and HK056-002, and Urelumab antibodies as positive controls) were added in gradient dilution at the same time as the cells, as shown in FIG. 6. The amount of IFN-. Gamma.expressed in the supernatant was measured after incubation at 37℃for 3 days.

The results are shown in FIG. 6, in which bispecific antibodies induced secretion of IFN-gamma by PBMC only in the presence of B7H3, as compared to negative control IgG.

Example 8 reporter Gene detection of bispecific antibody-mediated ADCC Effect on the B7H3 terminal

Jurkat/FcgammaRIIIa (158V)/luc cells (Promega) were cultured to log phase, digested, resuspended, counted and added to 96 well plates at 3X 10 ⁴ cells per well, MCF-7 cells were cultured to log phase, digested, resuspended, counted and added to corresponding 96 well plates at 3X 10 ⁴ cells per well. Samples to be tested (bispecific antibodies HK056-001, HK056-002, anti-B7H3, HK056-001 mock and IgG) were diluted to 60nM, 4-fold gradient diluted 7 spots, added to 96-well plates, incubated in a 37℃incubator for 18H, 100. Mu.l ONE-Glo Luciferase ASSAY SYSTEM reagent (Promega) was added to each well, and after incubation at room temperature for 10min, the chemiluminescent values were detected.

As shown in FIG. 7, the IgG4 subtype bispecific antibody HK056-002 had no ADCC effect on MCF-7 cells, and HK056-001 had significant ADCC effect in the presence of MCF-7.

Example 9 detection of bispecific antibody mediated ADCC killing of tumor cells by human PBMC

PBMCs were isolated from fresh blood of healthy humans using lymphocyte separation fluid (Sigma) following the protocol described, incubated overnight at 37 ℃. Samples to be tested (bispecific antibodies HK056-001, HK056-002, anti-B7H3, HK056-001 mock and IgG) were diluted to 5nM, 10-fold gradient diluted 5 spots, respectively, and added to 96-well plates. MCF-7, SKOV3 and PC-3 cells were cultured to the logarithmic growth phase, and after digestion and counting, they were labeled with CALCEIN AM (manufacturer YEASEN, cat. No. 40719ES 80), and the labeled target cells were added to 96-well plates, 1X 10 ⁴ cells per well, and incubated in a 37℃incubator in the dark for 30min. PBMC cells incubated overnight in incubator were removed, washed once with 1640 medium, counted after resuspension, and 3 x 10 ⁵ PBMCs were added to each well in 96-well plates. After centrifugation at 100g for 2min, the culture was incubated in a 37℃incubator at 1000rpm for 5min in the absence of light, and the supernatant was placed in another 96-well plate and assayed for fluorescence by bioteck microplate reader. Cell death rate (%) = (fluorescence value of dosing group-SR)/(MR-SR) was calculated as follows. Wherein MR is the fluorescence value of the maximum release well, SR is the fluorescence value of the spontaneous release well, both groups have only labeled target cells, no effector cell PBMC is added, wherein 10% Triton X-100 is added 30 minutes before plate reading and culture is continued for the rest 30 minutes in a 37 ℃ incubator, the cells of the spontaneous release well do not undergo any treatment, and the supernatant is directly taken after final centrifugation to read the fluorescence value.

As shown in FIG. 8, HK056-001 had the ability to mediate the killing of tumor cells by ADCC effect of PBMC, slightly weaker than anti-B7H3, and HK056-002 did not have ADCC killing.

Example 10 detection of bispecific antibody mediated direct killing of tumor cells by human PBMC

Experiments of target cell-dependent PBMC killing tumor cells were used to evaluate the effect of bispecific antibodies on the activation of PBMC killing tumor cells in the presence of B7H3 antigen expressed by target cells, the target cells used being MCF-7 tumor cell line and CHO-K1 Chinese hamster ovary cancer cell line, and the effector cells used being PBMC.

Whole blood was collected from healthy persons and PBMCs were collected using lymphocyte isolates (Sigma) according to the instructions. CD3 antibodies (Biolegend, cat. 317325) were diluted to 800ng/ml or 80ng/ml with PBS, added to 96-well plates and incubated at 37℃for 2h. After removing the supernatant and washing with PBS, MCF-7 or CHO-K1 and PBMC are added to 96-well plates in a ratio of effector cells to target cells (abbreviated as "target ratio") of 3:1 or 1:3, wherein the target cells are 1X 10 ⁴ per well, and the different target ratios regulate the effector cells. The test antibodies (bispecific antibodies HK056-001 and HK056-002, and Urelumab antibodies as positive controls) were added in gradient dilution at the same time as the cells, as shown in FIG. 9. After incubation at 37 ℃ for 3 days, the viability of the tumor cells was measured with CCK-8 reagent (eastern kernel chemistry) and the killing rate of PBMCs on tumor cells was calculated.

As shown in FIG. 9, both HK056-001 and HK056-002 showed significant killing effect on B7H3 expressing MCF-7 tumor cell lines, but not B7H3 negative CHO-K1 cells.

Example 11 reporter Gene detection of activation of 4-1BB Signal pathway by bispecific antibody

The construction method of HEK293/4-1BB/NFKB-Luc cells was as follows, the recombinant plasmid pCDNA3.4-4-1BB prepared in example 5 was introduced into HEK293 cells (Shanghai cell bank of the national institute) together with plasmid B (pNF- κB-Luc, ubao organism, product number VT 1588) having the element sequence of NF κB and with Lipofectamine 3000 transfection reagent (Invitrogen). HEK293/4-1BB/NFkB-luc was obtained by pressure screening.

HEK293/4-1BB/NFkB-luc cells were cultured to logarithmic phase, digested, resuspended and counted and then added to 96-well plates with 3X 10 ⁴ cells per well, MCF-7 cells were cultured to logarithmic phase, digested, resuspended and counted and then added to corresponding 96-well plates with 3X 10 ⁴ cells per well. Samples to be tested (bispecific antibody HK056-001, HK056-002, anti-B7H2+anti-B7H 3, HK056-001 mock and IgG) were diluted to 60nM (anti-B7H2+anti-B7H 3, 60nM concentration of both monoclonal antibodies) respectively, diluted 7 spots in a 4-fold gradient, added to 96-well plates, incubated in a 37℃incubator for 18H, 100 μl ONE-Glo Luciferase ASSAY SYSTEM reagent (Promega) was added per well, incubated at room temperature for 10min, and chemiluminescent values were detected.

As a result, as shown in FIG. 10, both HK056-001 and HK056-002 were able to effectively activate the 4-1BB signaling pathway, whereas the combination of two parent monoclonal antibodies alone failed to activate the downstream 4-1BB signaling pathway.

EXAMPLE 12 in vivo efficacy detection of the inhibitory Effect of bispecific antibodies on CT26/HuB7H3 tumor growth CT26/HuB7H3 was constructed by artificially synthesizing the sequence of interest encoding the full length of human B7H3 (Uniprot#Q5ZPR 3) (the nucleotide sequence encoding HuB7H3 in Table 4). Then, the small fragment between the XbaI and HindIII cleavage recognition sites of the pCDNA3.4 vector was replaced with the target sequence according to the general method in the art to obtain recombinant plasmid pCDNA3.4-B7H3, and the obtained recombinant plasmid pCDNA3.4-B7H3 was introduced into wild CT26 cells by using Lipofectamine3000 transfection reagent (Invitrogen) after the correctness of the sequencing verification, thus obtaining cell strain CT26/HuB7H3 highly expressing human B7H3.

CT26/HuB7H3 cells were cultured in vitro, digested with pancreatin, and then inoculated subcutaneously with 1X 10 ⁶/Balb/c-H4-1 BB mice (Jiangsu Jiukang Biotech Co., ltd., strain No. T003359) of 6-8 weeks old, and after 6 days of inoculation, group administration was started (tumor average volume: about 72mm ³), 5 animals per group were administered intraperitoneally (each mouse administration volume: 100. Mu.l), twice a week for a total of 6 times. The dosing groups and dosing amounts were as follows:

1) Physiological saline;

2)HK056-001,2mg/kg;

3)HK056-001,8mg/kg;

4)HK056-002,8mg/kg;

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

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

7)mPD-1,5mg/kg。

Wherein mPD-1 is an anti-mouse PD-1 antibody (manufacturer: bio X cell, cat# BE0146-100 MG).

Animals were dosed twice weekly and checked for survival and activity including tumor growth, body weight, activity, diet and recorded. The calculated volume formula for tumor volume is 1/2×length×width×width (mm ³).

As shown in FIG. 11, HK056-001 has good tumor inhibition effect at 2mg/kg and 8mg/kg, 1 and 2 mice have complete tumor disappearance (CR) respectively, the drug effect is superior to that of the 8mg/kg dose group of HK056-002 and the combined group of two parent monoclonal antibodies, and the combined group shows better anti-tumor effect when combined with PD-1 antibodies, and 4 mice have complete tumor disappearance.

Example 13 challenge experiments with CT26/HuB7H3 tumor models

The challenge experiment was performed after further observation of the Cured (CR) mice in example 12 for at least one month without recurrence of the tumor, after digestion with pancreatin, the Cured (CR) Balb/c-h4-1BB mice were inoculated subcutaneously according to 1.25X10 ⁶/day, and after inoculation for 39 days, no tumor growth was observed, indicating that the treated mice had immunological memory and that tumors did not recur after challenge. The experimental results are shown in FIG. 12.

EXAMPLE 14 in vivo efficacy detection of the inhibitory Effect of bispecific antibodies on MC38/HuB7H3 tumor growth

Construction of MC38/HuB7H3 cell line the pCDNA3.4-B7H3 plasmid was synthesized as described in example 12 and introduced into wild-type MC38 cells using Lipofectamine 3000 transfection reagent (Invitrogen Co.) to obtain MC38/HuB7H3 cell line highly expressing human B7H3.

MC38/HuB7H3 cells were cultured in vitro, digested with pancreatin, and then subcutaneously inoculated in 1X 10 ⁶/B6-hCD 137 mice (Jiangsu Jiugai Kangsu Biotech Co., ltd., strain No. T003360) of 8-9 weeks old, and 7 days after the inoculation, group administration was started (tumor average volume: about 52mm ³), 6 animals per group were administered intraperitoneally (each mouse administration volume: 100. Mu.l), and twice weekly for a total of 5 times. The dosing groups and dosing amounts were as follows:

1) Physiological saline;

2)HK056-001,3mg/kg;

3)HK056-001,1mg/kg;

4)HK056-001,0.3mg/kg;

as shown in FIG. 13, HK056-001 showed significant dose-dependent antitumor activity in 4-1BB humanized mice vaccinated with MC38-HuB7H 3.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

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 as positions 1-118 of SEQ ID No.1; and 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. 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 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. 5. The bispecific antibody according to claim 4, characterized in that the amino acid sequence of the 4-1BB antigen binding domain is as 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 as SEQ ID No. 1, and the amino acid sequence of the other chain is shown as 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 as 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 as 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.