Anti-b7h3-met dual-target nanobody drug conjugates and uses thereof

Abstract

The present application relates to anti-B7H3-MET dual-target nanobody drug conjugates and uses thereof. Specifically, the present application provides a dual-specific antibody containing B7H3 and c-MET nanobodies and a drug conjugate ADC thereof. The dual-specific antibody of the present application has high specificity and high endocytosis activity, and after conjugation with a toxin, a DAR-distributed uniform ADC is prepared. The ADC of the present application exhibits excellent killing effect on a variety of tumor cells in in vitro and in vivo experiments, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to anti-B7H3-MET dual-target nanobody drug conjugates and their uses. Background Technology

[0002] B7-H3, also known as CD276, is a type I transmembrane protein belonging to the B7-CD28 superfamily. Structurally similar to PD-L1, B7 family molecules can provide stimulatory signals to enhance and maintain T-cell immune responses, or produce inhibitory signals to limit and weaken T-cell immune responses. B7-H3 is widely expressed in various tumor tissues and has immunosuppressive effects in the tumor microenvironment (TME), closely associated with poor prognosis. High expression levels are associated with decreased overall survival (OS) and disease-free survival (DFS) in various cancers.

[0003] c-MET is a ligand of hepatocyte growth factor (HGF). Under normal circumstances, the binding of HGF and c-MET can mediate embryogenesis, tissue regeneration, wound healing, and the formation of nerves and muscles. However, in cancer cells, abnormal activation of the HGF / c-MET pathway (closely related to c-MET gene mutations, overexpression, and amplification) can stimulate many downstream signaling pathways such as PI3K / AKT, Ras / MAPK, JAK / STAT, SRC, and Wnt / β-catenin, thereby promoting tumor formation, invasive growth, and metastasis.

[0004] Currently, treatment options for tumors mediated by B7H3 or c-MET targets include radioimmunotherapy, small molecule kinase inhibitors, CAR-T therapy, monoclonal antibodies, and antibody-drug conjugates (ADCs). Several single-target drugs have entered the clinical stage, but their indications are limited to lung cancer, and research on other tumor indications is still in its early stages.

[0005] Bioinformatics analysis and translational studies have shown that cMET and B7H3 are co-expressed in a variety of solid tumors, but not highly expressed in normal tissues. Therefore, simultaneously targeting B7H3 and cMET may become a new and powerful strategy in pan-cancer therapy.

[0006] In order to overcome the existing technological limitations of single-target ADC drugs and expand the potential treatment population and tumor types, there is an urgent need in the field to develop dual-target drugs that target B7-H3 and c-MET. Summary of the Invention

[0007] The purpose of this invention is to provide a bispecific antibody targeting B7-H3 and c-MET, a dual-target ADC, and their application in the treatment of tumors.

[0008] In a first aspect of the present invention, a bispecific antibody is provided, characterized in that the bispecific antibody comprises: A first antigen-binding domain D1, wherein D1 includes an antibody or an antigen-binding fragment thereof targeting the B7-H3 protein; and The second antigen-binding domain D2 includes an antibody or an antigen-binding fragment thereof that targets the c-MET protein; The antibody or its antigen-binding fragment targeting the B7-H3 protein has the following CDR region: CDR1 shown in SEQ ID NO: 1 CDR2 shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3.

[0009] In another preferred embodiment, the CDR sequence is partitioned based on the Kabat rule.

[0010] In another preferred embodiment, the antibody includes: VHH, single-chain antibody scFv, double-chain antibody, monoclonal antibody, chimeric antibody, murine antibody, and humanized antibody.

[0011] In another preferred embodiment, the antigen-binding fragment comprises a Fab fragment, an F(ab')2 fragment, and an Fv fragment.

[0012] In another preferred embodiment, D1 comprises one or more VHHs that target the B7-H3 protein.

[0013] In another preferred embodiment, the D2 comprises one or more VHHs that target the c-MET protein.

[0014] In another preferred embodiment, D1 is VHH targeting the B7-H3 protein.

[0015] In another preferred embodiment, the VHH further includes a frame region (FR).

[0016] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0017] In another preferred embodiment, the VHH targeting the B7-H3 protein has an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to the sequence shown in SEQ ID NO: 4.

[0018] In another preferred embodiment, the amino acid sequence of the VHH targeting the B7-H3 protein contains one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 4, preferably conserved amino acid substitutions.

[0019] In another preferred embodiment, the amino acid sequence of the VHH targeting the B7-H3 protein is shown in SEQ ID NO: 4.

[0020] In another preferred embodiment, the antibody targeting the c-MET protein or its antigen-binding fragment has the following CDR region: CDR1 shown in SEQ ID NO: 5 CDR2 shown in SEQ ID NO: 6, and CDR3 as shown in SEQ ID NO: 7.

[0021] In another preferred embodiment, the CDR sequence is partitioned based on the Kabat rule.

[0022] In another preferred embodiment, D2 is a VHH that targets the c-MET protein.

[0023] In another preferred embodiment, the VHH further includes a frame region (FR).

[0024] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0025] In another preferred embodiment, the VHH targeting the c-MET protein has an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to the sequence shown in SEQ ID NO: 8.

[0026] In another preferred embodiment, the amino acid sequence of the VHH targeting the c-MET protein contains one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 8, preferably conserved amino acid substitutions.

[0027] In another preferred embodiment, the amino acid sequence of the VHH targeting the c-MET protein is shown in SEQ ID NO: 8.

[0028] In another preferred embodiment, the bispecific antibody further comprises the Fc segment of an immunoglobulin.

[0029] In another preferred embodiment, the immunoglobulin is IgG, including IgG1, IgG2, IgG3 or IgG4; preferably IgG1; more preferably human IgG1.

[0030] In another preferred embodiment, the Fc segment of the IgG1 contains a combination of knock-in-hole mutations.

[0031] In another preferred embodiment, the knob-into-hole mutation combination includes a knob mutation and a mortar mutation, wherein the knob mutation comprises T366W and the mortar mutation comprises T366S / L368A / Y407V.

[0032] In another preferred embodiment, the bispecific antibody has the structure shown in Formula I from the N-terminus to the C-terminus: (I) in, V1 is VHH that targets the B7-H3 protein; V2 is a VHH that targets the c-MET protein; L1 and L2 are each independently a non-connected peptide or hinge region; Fc1 and Fc2 are each independently the Fc fragment of an immunoglobulin; "‖" represents a disulfide bond or a knot-into-hole connection; "-" represents a peptide bond.

[0033] In another preferred embodiment, L1 and L2 are each independent of each other.

[0034] In another preferred embodiment, the linker peptide is a flexible linker peptide.

[0035] In another preferred embodiment, the linker peptide is represented by (G4S)n, where n is an integer selected from 1 to 6, preferably 2, 3, 4 or 5.

[0036] In another preferred embodiment, the hinge region is an IgG hinge region.

[0037] In another preferred embodiment, the bispecific antibody is a heterodimer.

[0038] In another preferred embodiment, the bispecific antibody comprises two single chains, wherein the first single chain comprises a VHH targeting the B7-H3 protein and an Fc segment targeting IgG1, and the second single chain comprises a VHH targeting the c-MET protein and an Fc segment targeting IgG1.

[0039] In another preferred embodiment, the VHH targeting the B7-H3 protein and the Fc segment of IgG1 are directly linked (without linkers).

[0040] In another preferred embodiment, the VHH of the target c-MET protein and the Fc segment of IgG1 are directly linked (without linkers).

[0041] In another preferred embodiment, the sequence of the bispecific antibody is selected from the group consisting of: (1) The amino acid sequence of the first single chain is shown in SEQ ID NO: 9, and the amino acid sequence of the second single chain is shown in SEQ ID NO: 10; (2) has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to the sequence shown in (1), and has a polypeptide sequence that simultaneously binds B7-H3 activity and anti-c-MET activity; (3) A polypeptide derived from (1), wherein the derivation refers to a sequence formed by substitution, deletion or addition of one or more amino acid residues, and has a polypeptide sequence having both B7-H3 binding activity and anti-c-MET activity.

[0042] In another preferred embodiment, the sequence of the bispecific antibody is as follows: the amino acid sequence of the first single chain is shown in SEQ ID NO: 9, and the amino acid sequence of the second single chain is shown in SEQ ID NO: 10.

[0043] In a second aspect of the invention, a polynucleotide or combination of polynucleotides is provided, said polynucleotide or combination of polynucleotides encoding a bispecific antibody as described in the first aspect of the invention.

[0044] In another preferred embodiment, the polynucleotide is selected from DNA, RNA, or a combination thereof.

[0045] In another preferred embodiment, the polynucleotide combination comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first single strand and the second polynucleotide encodes a second single strand; the amino acid sequence of the first single strand is shown in SEQ ID NO: 9 and the amino acid sequence of the second single strand is shown in SEQ ID NO: 10.

[0046] In a third aspect of the invention, an expression vector is provided, the expression vector containing a polynucleotide molecule as described in the second aspect of the invention.

[0047] In another preferred embodiment, the expression vector is a virus or plasmid, preferably a bacteriophage or bacteriophage particle.

[0048] In another preferred embodiment, the expression vector is selected from the group consisting of: pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0.

[0049] In a fourth aspect of the invention, a host cell is provided, the host cell containing the expression vector as described in the third aspect of the invention.

[0050] In another preferred embodiment, the host cell is selected from the group consisting of COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3) or TG1, more preferably E. coli TG1 or BL21(DE3) cells.

[0051] In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell or a 293F cell.

[0052] In a fifth aspect of the invention, a method for preparing a bispecific antibody as described in the first aspect of the invention is provided, the method comprising the following steps: a) Under expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express bispecific antibodies; b) Isolate and purify the bispecific antibody described in step a).

[0053] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising: (a) the bispecific antibody as described in the first aspect of the invention; and (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0054] In another preferred embodiment, the bispecific antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0055] In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

[0056] In another preferred embodiment, the coupling portion is a drug or toxin.

[0057] In another preferred embodiment, the drug is selected from the group consisting of: topoisomerase (Topo) inhibitors, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, vinca alkaloids, or derivatives thereof, or combinations thereof.

[0058] In another preferred embodiment, the toxin is selected from the group consisting of: camptothecin derivatives, MMAE, MMAF, chlortetracycline, phenytoin, pyrethroid, pyrethroid A-chain, cobustatin, docalimicin, doralastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, or derivatives thereof, or combinations thereof.

[0059] In another preferred embodiment, the chemical formula of the toxin is shown in Formula A: (Formula A).

[0060] In another preferred embodiment, in the antibody-drug conjugate (ADC), the bispecific antibody is linked to a drug or toxin via a connector.

[0061] In another preferred embodiment, the linker is selected from one or more: maleimide hexanoyl (MC), maleimide (MAL), succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC) linkers are connected to the antibody moiety and contain valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0062] In another preferred embodiment, the coupling portion is a detectable marker.

[0063] In another preferred embodiment, the coupling portion is a chemical marker and a biological marker.

[0064] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0065] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0066] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising: (i) an effective amount of a bispecific antibody as described in the first aspect of the invention, or an immunoconjugate as described in the sixth aspect of the invention, and (ii) a pharmaceutically acceptable carrier.

[0067] In another preferred embodiment, the pharmaceutical composition further comprises other bioactive substances, such as drugs for treating tumors.

[0068] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral / nasal spray and nebulized inhalation.

[0069] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid, solid, or gel.

[0070] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0071] In another preferred embodiment, the pharmaceutical composition is an injection.

[0072] In an eighth aspect of the invention, the use of a bispecific antibody as described in the first aspect of the invention, or an immunoconjugate as described in the sixth aspect of the invention, in the preparation of a medicament for treating tumors is provided.

[0073] In another preferred embodiment, the tumor is a tumor that highly expresses B7-H3 and / or c-MET.

[0074] In another preferred embodiment, the tumors include: colorectal cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), gastric cancer, oral cancer, liver cancer, breast cancer, ovarian cancer, neuroblastoma, pancreatic cancer, renal cell carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, retinoblastoma, schwannoma, glioma, and sarcoma.

[0075] In another preferred embodiment, the tumor is colorectal cancer.

[0076] In a ninth aspect of the invention, a method for treating a tumor is provided, the method comprising administering to a subject in need a bispecific antibody as described in the first aspect of the invention and an immunoconjugate as described in the sixth aspect of the invention.

[0077] In another preferred embodiment, the method further includes administration in combination with other drugs for treating tumors.

[0078] In another preferred embodiment, the tumor is defined as described in the eighth aspect of the invention.

[0079] In another preferred embodiment, the subjects in need include humans with tumors or non-human mammals such as mice, rats, or monkeys.

[0080] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0081] Figure 1 The diagram shows the structure and antigen-binding activity of the B7H3-MET dual-target nanobody; A is the structural diagram; B is the binding activity with human B7H3-ISO1 antigen; and C is the binding activity with human MET-ECD antigen.

[0082] Figure 2 The protein expression levels of B7H3 and MET in six types of colorectal cancer cells are shown; A is the Western blot result; B is the quantitative data of the relative expression level of B7H3 in A; C is the quantitative data of the relative expression level of MET in A.

[0083] Figure 3The endocytosis rates of BM04 and the control antibody in colorectal cancer cells are shown; A represents the endocytosis rate in HT29 cells; B represents the endocytosis rate in SW480 cells.

[0084] Figure 4 The following are the HIC diagrams for BM04 conjugated with different drugs: A is the HIC diagram for conjugated MMAE (i.e., BM04-MMEA); B is the HIC diagram for conjugated MY78 (i.e., BM04-MY78).

[0085] Figure 5 The killing effects of BM04-MMAE on different tumor cell types are shown. A represents the killing effect on COLO320 cells; B represents the killing effect on HCT116 cells; C represents the killing effect on HT29 cells; D represents the killing effect on LS1034 cells; E represents the killing effect on RKO cells; and F represents the killing effect on SW480 cells.

[0086] Figure 6 The killing effect of BM04-MY78 in different tumor cells is shown; A shows the killing effect in HT29 cells; B shows the killing effect in HCT116 cells.

[0087] Figure 7 The effects of BM04-MMAE on mice with the NCI-H1975 tumor-bearing model are shown; A represents the change in mouse body weight; B represents the change in tumor volume.

[0088] Figure 8 The effects of BM04-MMAE on the HT29 tumor-bearing mouse model are shown; A represents the change in mouse body weight; B represents the change in tumor volume.

[0089] Figure 9 The effects of BM04-MY78 on mice with a HT29 tumor-bearing model are shown; A represents the change in mouse body weight; B represents the change in tumor volume.

[0090] Figure 10 The effects of BM04-MY78 on mice with a HCT116 tumor-bearing model are shown; A represents the change in mouse body weight; B represents the change in tumor volume. Detailed Implementation

[0091] Through extensive and in-depth research, the inventors have developed a B7H3-MET dual-target nanobody and its drug conjugate. This invention's bispecific antibody simultaneously targets both B7H3 and MET, and the nanobody possesses the characteristics of small molecular weight and rapid penetration into tumor tissue, exhibiting better toxin delivery capability compared to traditional antibodies. The bispecific antibody of this invention exhibits high specificity and high endocytic activity, and the ADC prepared after conjugation with a toxin shows uniform DAR distribution. The ADC of this invention demonstrates excellent killing effects against various tumor cells in in vitro and in vivo experiments. Based on these findings, this invention was completed.

[0092] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0093] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0094] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0095] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0096] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0097] The "immunoglobulin antibody IgG" described in this invention is a molecule of approximately 150 kDa, composed of four peptide chains. It contains two identical γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus exhibiting a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and each is linked to a light chain. The resulting tetramer has two identical halves, forming a fork-like or Y-like shape, with each end of the fork containing an identical antigen-binding site. IgG antibodies can be classified into several subclasses (e.g., IgG1, 2, 3, 4) based on minor differences in the amino acid sequence of the constant region of the heavy chain.

[0098] In this invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0099] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0100] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that has antigen-binding activity. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fv fragments; or (iv) single-chain Fv (scFv). As used herein, the term "antigen-binding fragment" also encompasses other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, bisomal antibodies, trisomal antibodies, tetrasomal antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0101] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0102] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can be represented as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0103] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the frame regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0104] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin. Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in a linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0105] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 An antibody binds to an antigen with an equilibrium dissociation constant (KD) of M or less. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using an ELISA to determine the relative affinity of antibody-antigen binding.

[0106] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0107] B7-H3 Human B7-H3 is located on chromosome 15 and has two subtypes: 2IgB7-H3 and 4IgB7-H3. 2Ig-B7-H3 consists of 306 amino acids and has a single IgV-IgC pair; currently, there is no evidence that it participates in human tumor immunity. 4Ig-B7-H3 (the major human form) consists of 534 amino acids and contains two IgV-IgC pairs. This is the major form and is closely related to tumor immune escape, exerting its immune function solely through receptor binding. B7-H3 has three main expression forms: membrane-bound, soluble, and exosome-associated. The membrane-bound form is expressed on the surface of tumor cells, antigen-presenting cells (APCs), and immune cells, and is also present in cytoplasmic vesicles and the nucleus; it is the main form in which it exerts its immune regulatory and tumor-related functions.

[0108] B7-H3 is widely expressed in various tumor tissues, including gastric cancer (GC), oral squamous cell carcinoma (OSCC), hepatocellular carcinoma (HCC), breast cancer (BC), ovarian cancer (OC), and non-small cell lung cancer (NSCLC). The PI3K / AKT / mTOR (ILT4 activation), mTORC1 / YY2 (phosphorylated transcription factor YY2), JAK2 / STAT3, and p38 / MAPK-eIF4E pathways all positively regulate B7-H3 expression.

[0109] B7-H3 drives tumor proliferation and promotes tumor migration and invasion. For example, it inhibits apoptosis (downregulates CDK4, CDK6, and cyclin-D1) and upregulates the proliferation marker phosphorylated AKT, leading to abnormal cell cycle, by activating oncogenic pathways such as ERK, PI3K, and STAT3. Through pathways such as JAK2 / STAT3 / Slug, PI3K / AKT, and p38 / ERK, it upregulates EMT markers such as MMP-2, vimentin, and N-cadherin, downregulates E-cadherin, and induces epithelial-mesenchymal transition (EMT), participating in tumor cell proliferation and metastasis. It also creates favorable conditions for tumor growth by regulating the immunosuppressive state of the tumor microenvironment.

[0110] c-MET The human c-MET gene is located on chromosome 7 (7Q21-Q31), comprising 21 exons and 20 introns, encoding a protein of approximately 120 kDa. Its extracellular domain consists of: 1. an α-chain (32 kDa): containing a SEMA domain (mediating HGF binding and receptor dimerization), a PSI hinge region (involved in protein maturation), and an IPT1 domain (assisting HGF binding); 2. a β-chain (120 kDa): containing a transmembrane region connecting extracellular and intracellular structures. Its intracellular domain consists of: 1. a juxtamembrane domain (JM): containing the tyrosine residue Y1003, regulating MET endocytosis and degradation; 2. a tyrosine kinase domain: containing activation sites Y1234 and Y1235, which, upon phosphorylation, activate downstream signaling; and 3. a C-terminal docking site: containing phosphorylation sites Y1349 and Y1356, recruiting adaptor proteins (such as GAB1 and GRB2) to initiate signaling cascades.

[0111] The carcinogenic effects of c-MET include: 1. Synergistic effect with the TGF-β pathway to jointly promote EMT and M2 macrophage polarization and inhibit CD8+ T cell infiltration, such as forming an "immune desert" microenvironment in pancreatic cancer; 2. Cross-activation with the EGFR pathway: In EGFR-TKI resistant NSCLC, MET amplification can bypass EGFR inhibition through the PI3K / AKT pathway, forming "compensatory activation"; 3. Interaction with the Galectin family: Gal-3, Gal-4, Gal-7, etc., can enhance tumor cell adhesion, migration and immune escape by interacting with c-MET, thus promoting tumor metastasis.

[0112] The bispecific antibody of the present invention As used herein, the terms "bispecific antibody of the present invention" and "anti-B7H3-MET dual-target nanobody" are used interchangeably, both referring to the antibody provided in the present invention that simultaneously targets B7H3 and c-MET, and having an antibody or antigen-binding fragment targeting B7-H3 and c-MET proteins. In a preferred embodiment, the antigen-binding domain of the bispecific antibody of the present invention is provided in the form of a nanobody VHH.

[0113] In a preferred embodiment, the bispecific antibody of the present invention comprises a VHH targeting B7-H3, which has the following CDR sequence: CDR1: TYAMA (SEQ ID NO: 1) CDR2: VVNWSGGSTYYADSVKG (SEQ ID NO: 2), and CDR3: GGAGSYYGGAEYDY (SEQ ID NO: 3).

[0114] In some embodiments, the VHH targeting B7-H3 of the present invention has the amino acid sequence shown in SEQ ID NO: 4: EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYAMAWFRQAPGKEREFVAVVNWSGGSTYYADSVKGRFTISRDKSKNSMWLQMDSLRPEDTAVYYCAAGGAGSYYGGAEYDYWGQGTQVTVSS (SEQ ID NO: 4).

[0115] In some embodiments, the VHH chain has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 4.

[0116] In a preferred embodiment, the bispecific antibody of the present invention comprises a VHH targeting c-MET, which has the following CDR sequence: CDR1:NYAMT (SEQ ID NO: 5) CDR2: GISSAGGSTTYADSVKG (SEQ ID NO: 6), and CDR3: DRWSGVGYPPRAN (SEQ ID NO: 7).

[0117] In some embodiments, the VHH targeting B7-H3 of the present invention has the amino acid sequence shown in SEQ ID NO: 8: EVQVVESGGGLVQPGGSLRLSCAASGFTFRNYAMTWVRQAPGKEPEWVSGISSAGGSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPDDTGVYYCAKDRWSGVGYPPRANWGQGTQVTVSS (SEQ ID NO: 8).

[0118] In some embodiments, the VHH chain has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence shown in SEQ ID NO: 8.

[0119] The bispecific antibody of the present invention is preferably a heterodimer comprising two single chains, wherein the first single chain comprises a VHH targeting B7-H3 protein and an Fc segment targeting IgG1, and the second single chain comprises a VHH targeting c-MET protein and an Fc segment targeting c-MET protein.

[0120] The Fc segment of the IgG1 preferably contains a combination of knock-in-hole mutations, including knock-in-hole and knock-in-hole mutations, wherein the knock-in-hole mutation includes T366W and the knock-in-hole mutation includes T366S / L368A / Y407V.

[0121] In a preferred embodiment, the amino acid sequence of the first single chain is shown in SEQ ID NO: 9: EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYAMAWFRQAPGKEREFVAVVNWSGGSTYYADSVKGRFTISRDKSKNSMWLQMDSLRPEDTAVYYCAAGGAGSYYGGAEYDYWGQGTQVTVSSEPGSADETHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) In a preferred embodiment, the amino acid sequence of the second single chain is shown in SEQ ID NO: 10: EVQVVESGGGLVQPGGSLRLSCAASGFTFRNYAMTWVRQAPGKEPEWVSGISSAGGSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPDDTGVYYCAKDRWSGVGYPPRANWGQGTQVTVSSEPGSADETHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0122] In this invention, the bispecific antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the bispecific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0123] Table 1 Nucleic acid encoding and expression vector In another aspect, the present invention provides a polynucleotide molecule encoding the aforementioned bispecific antibody. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0124] The method for preparing the nucleotide molecule described in this invention is a conventional method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR, or obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by artificial full-sequence synthesis.

[0125] Those skilled in the art will understand that the nucleotide sequence encoding the amino acid sequence of the aforementioned bispecific antibody can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases encoding the bispecific antibody gene within the range of maintaining antibody activity.

[0126] In another aspect, the present invention provides an expression vector containing the aforementioned nucleotide molecules. The expression vector is a conventional expression vector in the art, referring to an expression vector containing suitable regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other suitable sequences. The expression vector can be a virus or a plasmid, such as a suitable bacteriophage or phage particle.

[0127] The present invention also provides a host cell containing the above-described expression vector.

[0128] The host cells described in this invention are any conventional host cells in the art, as long as they can stably and spontaneously replicate the recombinant expression vector and effectively express the carried nucleotides. The host cells include prokaryotic expression cells and eukaryotic expression cells. Transforming the aforementioned expression vector into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation. As a preferred embodiment, the host cells are eukaryotic cells. CHO cells or 293F cells are preferred.

[0129] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0130] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0131] Another aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody that specifically binds to B7-H3 and c-MET, the method comprising the following steps: a) Under the expression conditions, the above-mentioned host cells are cultured to express bispecific antibodies that can specifically bind to B7-H3 and c-MET; b) Isolate and purify the bispecific antibody described in step a).

[0132] The host cell culture method and the antibody isolation and purification method described in this invention are conventional methods in the art. For specific operating procedures, please refer to the relevant cell culture technology manual and antibody isolation and purification technology manual. The method for preparing the bispecific antibody disclosed in this invention includes: culturing the aforementioned host cells under expression conditions to express a bispecific antibody that specifically binds to B7-H3 and c-MET; and isolating and purifying the bispecific antibody. Using the above method, recombinant proteins can be purified into a substantially homogeneous substance.

[0133] The anti-B7-H3 and c-MET bispecific antibodies disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer and pH adjustment can be used to elute the anti-B7-H3 and c-MET bispecific antibodies bound to the affinity column. The inventors of this invention conducted detection experiments on the obtained bispecific antibodies, and the experimental results showed that the anti-bispecific antibodies can bind well to target cells and antigens, exhibiting high affinity.

[0134] Immunoconjugates The present invention also provides immunoconjugates (ADCs) based on the bispecific antibodies of the present invention. Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0135] The bispecific antibody of the present invention can be coupled to the effector molecule via a coupling agent. Examples of the coupling agent may be any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide-based coupling agents. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent may be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0136] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0137] Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0138] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0139] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0140] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0141] It should be understood that the drug in the ADC of the present invention comprises derivatives of the above-mentioned drug categories, such as camptothecin derivatives. In some embodiments, the drug in the ADC of the present invention is a camptothecin derivative as described in CN 121449623 A. In a preferred embodiment, the drug in the ADC of the present invention is MY78, which has the structure shown in Formula A: (Formula A) In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0142] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0143] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0144] In some embodiments, the method of the present invention includes binding an antibody to a adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0145] The ADC prepared from the bispecific antibody provided by this invention has a uniform drug-antibody ratio (DAR). In some embodiments, the drug-antibody ratio is in the range of 2 to 5, for example, 2 to 3 or 3 to 4.

[0146] Pharmaceutical Composition Another aspect of the present invention provides a composition comprising the above-described bispecific antibody or its pharmaceutical conjugate capable of specifically binding to B7-H3 and c-MET, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Preferably, the composition is a pharmaceutical composition.

[0147] The bispecific antibodies or their drug conjugates provided by this invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical formulations that exert therapeutic effects more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the bispecific antibodies of this invention, while also protecting the multifunctional groups of proteins from degradation (including but not limited to aggregation, deamination, or oxidation). Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH typically around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. Generally, liquid formulations are stable for at least one year at 2°C-8°C, and lyophilized formulations are stable for at least six months at 30°C. The bispecific antibody formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, and lyophilized formulations.

[0148] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described bispecific antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the bispecific antibody of the present invention can also be used with other therapeutic agents.

[0149] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0150] The beneficial effects of this invention include: 1. Nanobodies possess inherent advantages: Traditional antibodies have four polypeptide chains, including two identical heavy chains and two identical light chains, with a molecular weight of 150 kDa. Nanobodies have a smaller structural portion, containing only one heavy chain variable region, at only 15 kDa. Nanobodies fused with FC have a molecular weight of approximately 80 kDa, about half that of traditional antibodies. Therefore, nanobodies can recognize hidden antigen epitopes, exhibiting better binding specificity, tissue penetration, and low immunogenicity; simultaneously, nanobodies possess higher stability, are easier to modify and optimize, and are suitable for large-scale production.

[0151] 2. More Uniform DAR Distribution: The nanobody in this invention has two pairs of disulfide bonds between its two heavy chains. After toxin conjugation via disulfide bond reduction, the DAR distribution ranges from 0 to 4. Traditional antibody molecules have four pairs of disulfide bonds, and the DAR distribution ranges from 0 to 8 after toxin conjugation. Currently, the DAR values ​​of mainstream ADC products are generally around 4 / 8. For DAR4, this product is more uniform, and the proportion of DAR4 product is higher. The purity of the DAR4 target product based on traditional antibodies is mostly around 50%, while the purity of the DAR4 product in the ADC prepared by this invention is >90%.

[0152] 3. High Specificity and High Internalization Activity: The mechanism of action of ADC drugs is to specifically recognize tumor cell surface antigens through the targeting action of monoclonal antibodies. Target antigen-mediated internalization allows the ADC to enter the tumor cell, where it is hydrolyzed or enzymatically degraded to release the small molecule drug, thereby killing the tumor cells. Therefore, high specificity and high internalization activity are key factors for the efficacy of ADCs. This invention screened numerous B7H3 / cMET bispecific antibody candidates based on the principles of high specificity and high internalization activity. The screened B7H3 / cMET bispecific antibodies were then conjugated with toxins to obtain highly efficient B7H3 / cMET ADCs. The internalization rate of traditional antibodies in tumor cells is approximately 50%, while the internalization rate of the bispecific antibody containing the preferred nanobody of this invention is around 70%. The in vivo efficacy of the bispecific antibody ADC of this invention is significantly better than that of the control antibody ADC, demonstrating a synergistic effect of dual targets.

[0153] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0154] Example 1. Construction and binding activity (ELISA) detection of B7H3-MET dual-target nanobody In this embodiment, the B7H3-MET dual-target nanobody BMO4 was first constructed, and its structural schematic diagram is shown below. Figure 1 As shown in Part A, the sequences of the two single strands are shown as SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0155] The positive control antibodies used in this embodiment of the invention include: anti-B7-H3 antibody DS7300, whose sequences are SEQ ID NO: 6 and SEQ ID NO: 10 in patent CN103687945B; and anti-c-MET antibody ABT700, whose sequences are SEQ ID NO: 4 and SEQ ID NO: 10 in US Patent No. 8,741,290.

[0156] The binding affinity of the B7H3-MET dual-target nanobody BMO4 and the control antibody of this invention to human B7H3 antigen (B7H3-ISO1) and human MET antigen was tested. Specific experimental steps: 1. Coating antigen: Dilute the antigen to 1 μg / mL with PBS, add 100 μL of diluted antigen per well to the ELISA plate, and incubate overnight at 4 °C.

[0157] 2. Washing: Add 300 μL of 0.05% PBST to each well and wash twice.

[0158] 3. Blocking: Add 300 μL of PBS solution containing 3% BSA to each well and block at 37 °C for 2 h.

[0159] 4. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times.

[0160] 5. Primary antibody incubation: Add 100 μL of antibody dilution buffer to each well (dilute the antibody with PBS containing 1% BSA, with a maximum antibody concentration of 2 μg / mL, and perform 10-fold serial dilutions for a total of 4 concentrations), and incubate at room temperature for 1 hour.

[0161] 6. Washing: Add 300 μL of 0.05% PBST to each well and wash twice.

[0162] 7. Incubate the secondary antibody: Add 100 μL of secondary antibody (1:10000 dilution) to each well and incubate at 37 ℃ for 30 min.

[0163] 8. Washing: Add 300 μL of 0.05% PBST to each well and wash 4 times.

[0164] 9. Color development: Add 100 μL of TMB to each well and develop the color at 25 ℃ in the dark for 5-10 min.

[0165] 10. Termination: Add 50 μL of TMB colorimetric stop solution to each well to terminate the reaction.

[0166] 11. Detection: Use a TECAN microplate reader to read the plate at 450 nm and 630 nm and calculate the difference in absorbance.

[0167] 12. Data Processing: GraphPad Prism software was used to fit the antibody concentration and absorbance difference (OD450-OD630) to obtain a four-parameter fitting curve, and EC was calculated. 50 .

[0168] Experimental results are as follows Figure 1 As shown, the B7H3-MET dual-target nanobody BMO4 of the present invention can bind to human B7H3 antigen and human MET antigen, and its binding ability is comparable to that of single-target antibodies DS7300 and ABT700. The specific EC50 values ​​are shown in Table 2.

[0169] Table 2. EC50 (ELISA level) of BM04, DS7300, and ABT700 binding to antigens. Example 2. Detection of protein expression levels of MET and B7H3 in six types of colorectal cancer cells. In this embodiment, the protein expression levels of B7H3 and MET in six types of colorectal cancer cells were detected by Western blotting (WB). The specific experimental procedures are as follows: 1. Protein sample preparation: Remove cells, place on ice, centrifuge at 400g for 5 min at 4℃, wash twice with pre-cooled PBS and centrifuge to remove supernatant. Add RIPA lysis buffer to the cell pellet and vortex thoroughly to lyse. Quantify proteins using the BCA method.

[0170] 2. Electrophoresis: Use a 4-12% precast gel. Add 5 μL of protein marker and 18 μL of protein sample in sequence. Plug in the power cord and adjust the voltage to 80-90 V. After 15-30 min, when the bromophenol blue enters the lower gel, adjust the voltage to 120-130 V. Stop electrophoresis when the bromophenol blue just runs out of the gel.

[0171] 3. Membrane transfer: Take out the PVDF membrane and cut a piece with the same size and shape as the gel using scissors. Soak the cut PVDF membrane in methanol for 15 seconds before use for activation. After activation, the PVDF membrane completely covers the gel block. Remove the glass plate, cut the gel appropriately, place the gel on the membrane, and there should be no air bubbles between the membrane and the gel block. Then cover the membrane with filter paper and sponge soaked in transfer buffer, put the whole into the membrane transfer device, and then place it in the membrane transfer tank. Place the membrane side at the positive electrode and the gel side at the negative electrode. Put an ice box in the tank and fill it with transfer buffer. Adjust the voltage and set an appropriate membrane transfer time according to the molecular weight of the target protein for membrane transfer.

[0172] 4. Blocking: Wash the PVDF membrane with 0.1% TBST 2 - 3 times, 5 minutes each time, add 5% skim milk prepared, and block it on a shaker at room temperature for 60 minutes; 5. Washing: Rinse the blocked PVDF membrane with 0.1% TBST 3 times, 5 minutes each time; 6. Incubating primary antibody: Incubate overnight on a shaker at 4 °C; 7. Washing: Rinse the blocked PVDF membrane with 0.1% TBST 3 times, 5 minutes each time; 8. Incubating secondary antibody: Dilute the HRP - labeled secondary antibody corresponding to the species with 0.1% TBST and incubate on a shaker at room temperature for 60 minutes; 9. Washing: Rinse the NC membrane with 0.1% TBST 3 times, 10 minutes each time; 10. Color development: Mix solution A and solution B in the ECL luminescent agent in a 1:1 ratio and then drip it onto the PVDF membrane. Expose, develop, fix, and scan the film in a darkroom, and calculate the relative expression level through the gray value.

[0173] The results are as Figure 2 shown. Expression level of MET protein: SW480 < RKO < HT29 < LS1034 < HCT116 < COLO320. Expression level of B7H3 protein: SW480 < RKO < HT29 < HCT116 < LS1034 < COLO320.

[0174] Example 3. Endocytosis activity of B7H3 - MET bispecific nanobody In this example, the endocytosis activity of B7H3 - MET bispecific nanobody on colorectal cancer HT29 and SW480 cells was detected.

[0175] Specific experimental steps: 1. Plating: Digest the cells to be tested in the logarithmic growth phase with trypsin, centrifuge, collect the cells, resuspend the cells with PBS, and adjust the cell density to 3 10 6Cells / mL, add 100 μL of cell suspension to each well (96-well plate).

[0176] 2. Antibody incubation: Dilute B7H3 nanobody with culture medium (antibody concentration: 20 μg / mL), take 100 μL / well and add it to the well plate containing cell suspension, mix thoroughly, and incubate at 4 ℃ in the dark for 1 h.

[0177] 3. Washing: Centrifuge at 4 ℃ and 400 g for 5 minutes and discard the supernatant. Add 200 μL of complete culture medium to each well, centrifuge at 4 ℃ and 400 g for 5 minutes, discard the supernatant, and repeat twice.

[0178] 4. Plate separation: Add 200 μL of complete culture medium to each well, resuspend and mix the cells, transfer 100 μL / well to a new plate, and incubate at 37 ℃ for 2 h (endocytosis group). The remaining cell suspension is stored at 4 ℃ in the dark (control group).

[0179] 5. Washing: After incubation at 37 °C, the endocytic cells were centrifuged at 4 °C and 400 g for 5 minutes and the supernatant was discarded. 200 μL of PBS containing 1% FBS was added to each well, and the cells were centrifuged at 4 °C and 400 g for 5 minutes. The supernatant was discarded, and the process was repeated twice.

[0180] 6. Secondary antibody incubation: Add 100 μL of PE anti-hμman IgGFc Antibody dilution (1:100 dilution) to each well of the endocytosis group cells and the control group cells, and incubate at 4 ℃ in the dark for 30 min.

[0181] 7. Washing: After incubation, centrifuge at 400 g for 5 minutes at 4 ℃ and discard the supernatant. Add 200 μL of PBS containing 1% FBS to each well, centrifuge at 400 g for 5 minutes at 4 ℃, discard the supernatant, and repeat twice.

[0182] 8. Flow cytometry: Cells were reselected using PBS containing 100 μL / well of 1% FBS, and the median fluorescence intensity (Medium-PE) expressed in the test cells was detected using flow cytometry. Calculation: Internalization amount = Control cells (Medium-PE) - Internalized cells (Medium-PE), Internalization rate (%) = [1 - (Internalized cells Medium μm-PE) / (Control cells Medium-PE)] x 100% Experimental results are as follows Figure 3 As shown in the figure. Experimental results show that the B7H3-MET dual-target nanobody BM04 exhibits superior endocytosis effects compared to the control in colorectal cancer cells HT29 and SW480.

[0183] Example 4. B7H3-MET dual-target nanobody conjugated to MY78 In this embodiment, the anti-B7H3-MET dual-target nanobody was conjugated with the toxin VcMMAE or MY78 to prepare a B7H3-MET dual-target nanobody-drug conjugate for subsequent evaluation of tumor cell proliferation inhibition and in vivo antitumor activity. Specific experimental steps: 1. Antibody reduction With the total reduction volume and antibody mass fixed, calculate the amount of TCEP (10 eq, 10 times the amount of antibody) of reducing agent, the amount of EDTA-2Na (final concentration of 5 mM in the reduction system), and the volume of PBS buffer. Add PBS, EDTA-2Na, TCEP, and antibody in that order and mix. Reduce in a metal bath at 37 °C for 4 hours at a speed of 200 rpm.

[0184] 2. Couple with VC-MMAE or MY78 The feed equivalent of Vc-MMAE or MY78 was fixed at 10 eq. The feed volume was calculated based on the concentration of Vc-MMAE or MY78 stock solution. DMSO solvent was added to make the final concentration in the system 10%. After thorough mixing, the reduced solution was slowly added and reacted in a metal bath at 25°C for 2 hours at a speed of 200 rpm.

[0185] 3. Termination The reaction was terminated with L-Cysteine ​​solution, with an L-Cysteine ​​feed equivalent of 7 eq. The feed volume was calculated based on the stock solution concentration.

[0186] 4. Purification Centrifuge the conjugated solution at 8000 g for 5 min, discard the precipitate, and repeat 2-3 times. Select an appropriate ultrafiltration tube based on the total amount and volume of the nanobody-conjugated drug, transfer the supernatant to a 10 kDa ultrafiltration tube, and concentrate the sample by centrifuging at 3500-8000 g for 15 min. Then add His-HCl (10 mM, pH 6.0) containing 10% DMSO to the maximum mark, centrifuge at 3500-8000 g for 15 min, and repeat 4 times. Next, add His-HCl (10 mM, pH 6.0) to the maximum mark, centrifuge at 3500-8000 g for 15 min, and repeat 16 times. Remove the liquid from the ultrafiltration tube and determine the concentration and DAR value.

[0187] The results are as follows Figure 4 As shown. The purity of DAR4 target products based on traditional antibodies is mostly around 50%, while the purity of the BMO4-MMAE DAR4 product of this invention is >80% (e.g., Figure 4(Part A of the product), BM04-MY78 DAR4 has a purity of up to 99%.

[0188] Example 5. Detection of the cytotoxic activity of anti-B7H3-MET dual-target nanobody conjugated with MMAE product against tumor cells. In this embodiment, the VCMMAE conjugate of B7H3-MET nanobody was evaluated for its inhibitory activity on the proliferation of colorectal cancer cells RKO, HT29, SW480, HCT116, COLO320, and LS1034. Cell viability was detected using the CellTiter-Glo2.0 Cell Viability Assay.

[0189] Specific experimental steps: 1. Plating: Digest and collect the cells to be tested, and prepare a cell suspension (10,000 cells / mL) using complete culture medium. Add 100 μL of the cell suspension to each well of a 96-well black transparent flat-bottomed plate. Discard the wells at the edge of the plate and add 100 μL of PBS to each well. Place the plated cell plate in an incubator overnight to allow the cells to adhere.

[0190] 2. ADC incubation with cells: Dilute ADC with complete culture medium. Use 500 μg / mL for the first well, and then serially dilute 5-fold to obtain a total of 6 concentrations. Add 10 μL of ADC dilution to each well and incubate at 37 ℃ with 5% CO2 for 6 days.

[0191] 3. Detection: After incubation, add 100 μL / well of CellTiter-Glo 2.0Reagent detection solution to the 96-well cell culture plate, let stand for 15 min, and wait for the cells to completely lyse.

[0192] 4. Plate reading: The TECAN microplate reader detects the luminescence value of the test sample, and the survival rate is calculated based on the luminescence value to create a curve.

[0193] Experimental results are as follows Figure 5 As shown, BM04-MMAE exhibits strong in vitro killing ability in various tumor cell types in a dose-dependent manner, demonstrating broad-spectrum tumor suppression. In particular, its killing effect on HCT116 cells is superior to that of ABT700-MMAE and DS7300-MMAE.

[0194] Example 6. Detection of the cytotoxic activity of anti-B7H3-MET dual-target nanobody conjugated with MY78 product against tumor cells. In this embodiment, the inhibitory activity of the MY78 conjugate of the B7H3-MET nanobody on the proliferation of colorectal cancer cells HT29 and HCT116 was evaluated, and cell viability was detected using CCK8 assay.

[0195] Specific experimental steps: 1. Plating: Digest and collect the cells to be tested, and prepare a cell suspension (10,000 cells / mL) using complete culture medium. Add 100 μL of the cell suspension to each well of a 96-well black transparent flat-bottomed plate. Discard the wells at the edge of the plate and add 100 μL of PBS to each well. Place the plated cell plate in an incubator overnight to allow the cells to adhere.

[0196] 2. ADC incubation with cells: Dilute ADC with complete culture medium. Use 500 μg / mL for the first well, and then serially dilute 5-fold to obtain a total of 6 concentrations. Add 10 μL of ADC dilution to each well and incubate at 37 ℃ with 5% CO2 for 6 days.

[0197] 3. Detection: After incubation, add 10 μL of CCK8 detection solution to each well of a 96-well cell culture plate and incubate at 37°C for 2 h.

[0198] 4. Plate reading: TECAN microplate reader detects the OD450 of the sample and calculates the survival rate based on the value to create a curve.

[0199] Experimental results are as follows Figure 6 As shown, BM04-MY78 exhibits strong in vitro killing activity in both HT29 and HCT116. These results demonstrate that the B7H3-MET dual-target nanobody of this invention, when conjugated to different toxins, also possesses excellent killing activity.

[0200] Example 7. Pharmacodynamic evaluation of B7H3-MET dual-target nanobody-conjugated drug in immunodeficient mouse HCT116, HT29, and RKO xenograft models. 1. Antitumor efficacy of BM04-MMAE in a Balb / c Nude mouse xenograft RKO colorectal cancer tumor model Human colorectal cancer cells RKO (5×10^6 / 0.1mL / mouse (no Matrigel)) were inoculated into female Balb / c Nude mice (purchased from Vital River, 5-6 weeks old, 18-21g) and divided into 4 groups: solvent PBS group, BM04-MMAE, DS7300-MMAE, and ABT700-MMAE, with 5 mice in each group. The inoculation was performed subcutaneously in the right anterior flank, followed by a tail vein injection of ADC (2 mg / kg) or PBS. The mice were observed for 3 weeks. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and the time of drug administration was recorded.

[0201] Tumor volume (V) is calculated as (length × width) 2 ) / 2.

[0202] Tumor growth inhibition rate (TGI%) is calculated using the following formula: Tumor growth inhibition rate = (1 - change in tumor volume in the drug-treated group / change in tumor volume in the control group) × 100% Tumor volume results as follows Figure 7 As shown in Part B and Table 3, at a dose of 2 mg / kg on Day 23, tumor growth in the BM04-MMAE administration group was significantly inhibited, with a tumor inhibition rate of 67.16%, which was superior to the positive control ADC DS7300-MMAE (TGI 56.47%) and ABT-700-MMAE (TGI 51.46%).

[0203] Table 3. TGI on days 6, 13, and 23 of drug administration in the mouse NCI-H1975 tumor-bearing model. 2. Antitumor efficacy of BM04-MMAE against Balb / c Nude mouse xenografted HT29 colorectal cancer cell tumor model Female Balb / c Nude mice (purchased from Vital River, 5-6 weeks old, 18-21g) were subcutaneously inoculated with human colorectal cancer tumor cells HT29 (3.2×10^6 / 0.1mL / mouse (no Matrigel)) in the right anterior flank. The tumors were allowed to grow to 100-150mm. 3 At approximately 10:00 AM, mice were randomly divided into four groups: BM04-MMAE, DS7300-MMAE, and ABT700-MMAE, with five mice in each group. ADC (2 mg / kg) or PBS was administered via tail vein injection every two weeks for 17 days. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and the administration time was recorded.

[0204] Tumor volume (V) is calculated as (length × width) 2 ) / 2.

[0205] Tumor growth inhibition rate (TGI%) is calculated using the following formula: Tumor growth inhibition rate = (1 - change in tumor volume in the drug-treated group / change in tumor volume in the control group) × 100% like Figure 8 As shown in Part B and Table 4, administration of 2 mg / kg BM04-MMAE resulted in tumor regression in HT29 mice, demonstrating superior antitumor efficacy compared to the positive controls DS7300-MMAE and ABT-700-MMAE. BM04-MMAE had minimal impact on mouse body weight, and the weight of mice remained stable after administration (e.g., ...). Figure 8 (As shown in Part A).

[0206] Table 4. TGI on days 7, 14, and 17 of drug administration in the HT29 tumor-bearing mouse model. 3. Antitumor efficacy of BM04-MY78 against a Balb / c Nude mouse xenografted HT29 colorectal cancer cell tumor model Female Balb / c Nude mice (purchased from Vital River, 5-6 weeks old, 18-21g) were subcutaneously inoculated with human colorectal cancer tumor cells HT29 (4.2×10^6 / 0.1mL / mouse (no Matrigel)) in the right anterior flank. The tumors were allowed to grow to 100-200mm. 3 At approximately 21 weeks, mice were randomly divided into four groups: BM04-MY78, DS7300-Dxd, and ABT700-MMAE, with five mice in each group. ADC (5 mg / kg) or PBS was administered via tail vein injection every two weeks for 21 days. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and the administration time was recorded.

[0207] Tumor volume (V) is calculated as (length × width) 2 ) / 2.

[0208] Tumor growth inhibition rate (TGI%) is calculated using the following formula: Tumor growth inhibition rate = (1 - change in tumor volume in the drug-treated group / change in tumor volume in the control group) × 100% like Figure 9 As shown in Part B and Table 5, after administration of 5 mg / kg BM04-MY78 for 21 days, HT29 tumors regressed, and the antitumor effect was better than the positive control DS7300-MMAE, but slightly weaker than the same dose of ABT-700-MMAE.

[0209] Table 5. TGI on days 7, 14, and 21 of drug administration in the HT29 tumor-bearing mouse model. 4. Antitumor efficacy of BM04-MY78 against a Balb / c Nude mouse xenografted HCT116 colorectal cancer cell tumor model Female Balb / c Nude mice (purchased from Vital River, 5-6 weeks old, 18-21g) were subcutaneously inoculated with human colorectal cancer tumor cells HCT116 (5×10^6 / 0.1mL / mouse (no Matrigel)) in the right anterior flank. The tumors were allowed to grow to 100-200mm. 3At approximately 21 weeks, mice were randomly divided into four groups: BM04-MY78, DS7300-Dxd, and ABT700-MMAE, with five mice in each group. ADC (5 mg / kg) or PBS was administered via tail vein injection every two weeks for 21 days. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and the administration time was recorded.

[0210] Tumor volume (V) is calculated as (length × width) 2 ) / 2.

[0211] Tumor growth inhibition rate (TGI%) is calculated using the following formula: Tumor growth inhibition rate = (1 - change in tumor volume in the drug-treated group / change in tumor volume in the control group) × 100% like Figure 10 As shown in Part B and Table 6, after administration of 5 mg / kg BM04-MY78 for 21 days, HCT116 tumors regressed, and the anti-tumor effect was superior to the positive controls DS7300-MMAE and ABT-700-MMAE, demonstrating a significant anti-tumor effect.

[0212] Table 6. TGI on days 7, 14, and 21 after drug administration in a mouse HCT116 tumor-bearing model. All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody comprises: A first antigen-binding domain D1, wherein D1 includes an antibody or an antigen-binding fragment thereof targeting the B7-H3 protein; and The second antigen-binding domain D2 includes an antibody or an antigen-binding fragment thereof that targets the c-MET protein; The antibody or its antigen-binding fragment targeting the B7-H3 protein has the following CDR region: CDR1 shown in SEQ ID NO: 1 CDR2 shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO:

3.

2. The bispecific antibody as described in claim 1, characterized in that, D1 is a VHH targeting the B7-H3 protein; the amino acid sequence of the VHH targeting the B7-H3 protein is shown in SEQ ID NO:

4.

3. The bispecific antibody as described in claim 1, characterized in that, The antibody or its antigen-binding fragment targeting the c-MET protein has the following CDR region: CDR1 shown in SEQ ID NO: 5 CDR2 shown in SEQ ID NO: 6, and CDR3 as shown in SEQ ID NO:

7.

4. The bispecific antibody as described in claim 1, characterized in that, The bispecific antibody has the structure shown in Formula I from the N-terminus to the C-terminus: (I) in, V1 is VHH that targets the B7-H3 protein; V2 is a VHH that targets the c-MET protein; L1 and L2 are each independently a non-connected peptide or hinge region; Fc1 and Fc2 are each independently the Fc fragment of an immunoglobulin; "‖" represents a disulfide bond or a knot-into-hole connection; "-" represents a peptide bond.

5. The bispecific antibody as described in claim 1, characterized in that, The bispecific antibody comprises two single chains, wherein the first single chain contains the VHH and Fc segments of IgG1 targeting the B7-H3 protein, and the second single chain contains the VHH and Fc segments of IgG1 targeting the c-MET protein.

6. The bispecific antibody as described in claim 1, characterized in that, The sequence of the bispecific antibody is as follows: the amino acid sequence of the first single chain is shown in SEQ ID NO: 9, and the amino acid sequence of the second single chain is shown in SEQ ID NO:

10.

7. A polynucleotide or combination of polynucleotides, characterized in that, The polynucleotide or combination of polynucleotides encodes the bispecific antibody as described in any one of claims 1-6.

8. An expression carrier, characterized in that, The expression vector contains the polynucleotide or combination of polynucleotides as described in claim 7.

9. An immunoconjugate, characterized in that, The immunoconjugate includes: (a) The bispecific antibody as described in any one of claims 1-6; and (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

10. The immunoconjugate as described in claim 9, characterized in that, The coupling portion is a drug or toxin; The drug is selected from the group consisting of: topoisomerase (Topo) inhibitors, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, vinca alkaloids, or derivatives thereof, or combinations thereof; and / or The toxins are selected from the group consisting of: camptothecin derivatives, MMAE, MMAF, chlortetracycline, phenytoin, phenytoin A-chain, cobustatin, docalimcin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, or derivatives thereof, or combinations thereof.

11. The immunoconjugate as described in claim 10, characterized in that, The chemical formula of the toxin is shown in Formula A: (Formula A).

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) an effective amount of the bispecific antibody as described in any one of claims 1-6, or the immunoconjugate as described in any one of claims 9-11, and (ii) a pharmaceutically acceptable carrier.

13. Use of the bispecific antibody as described in any one of claims 1-6, or the immunoconjugate as described in any one of claims 9-11, in the preparation of a medicament for treating tumors; wherein the tumor is a tumor that highly expresses B7-H3 and / or c-MET, and the tumor comprises: Colorectal cancer, lung cancer, stomach cancer, oral cancer, liver cancer, breast cancer, ovarian cancer, neuroblastoma, pancreatic cancer, renal cell carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, retinoblastoma, schwannoma, glioma, sarcoma.

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