Bispecific antibodies against HER2 and their applications

Abstract

The present invention provides a bispecific antibody against HER2, which comprises a first protein domain and a second protein domain for recognizing extracellular domain II and extracellular domain IV of HER2, the first protein domain comprising a first heavy chain and a light chain, and the second protein domain comprising a second heavy chain and a light chain. The present invention also provides pharmaceutical compositions comprising the bispecific antibody and uses thereof. The bispecific antibody and pharmaceutical compositions comprising the same can be used to treat cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Chinese patent application No. 2019101090084, filed on February 3, 2019. The entire contents of the above Chinese patent application are incorporated herein by reference. [Technical field]

[0002] The present invention relates to the field of biopharmaceuticals, specifically to a bispecific antibody against HER2 and its applications. [Background technology]

[0003] HER2 is a 185 kDa cell surface receptor tyrosine kinase and a member of the epidermal growth factor receptor (EGFR) family, which includes four distinct receptors: EGFR / ErbB-1, HER2 / ErbB-2, HER3 / ErbB-3, and HER4 / ErbB-4. The four members of the EGFR family form homodimers and heterodimers, with HER2 being the preferred and strongest dimerization partner among the other ErbB receptors (Graus-Porta et al., Embo J 1997, 16:1647-1655; Tao et al., J Cell Sci 2008, 121:3207-3217). HER2 can be activated by overexpression or by heterodimerization with other ErbBs that can be activated by ligand binding (Riese and Stern, Bioessays 1998, 20:41-48). HER2 has no identified ligand. HER2 activation leads to receptor phosphorylation, which triggers downstream signaling cascades through multiple signaling pathways, such as MAPK, phosphoinositide 3-kinase / AKT, JAK / STAT, and PKC, ultimately regulating multiple cellular functions, such as proliferation, survival, and differentiation (Huang et al., Expert Opin Biol THER 2009, 9:97-110). Much of the interest in HER2 in oncology has focused on its role in breast cancer, where overexpression of HER2 has been reported in approximately 20% of cases and is associated with poor prognosis (Reese et al., Stem Cells 1997, 15:1-8; Andrechek et al., Proc Natl Acad Sci USA 2000, 97:3444-3449; and Slamon et al., Science 1987, 235:177-182). In addition to breast cancer, HER2 expression is associated with other human carcinoma types, including prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colon cancer, esophageal cancer, and squamous cell carcinoma of the head and neck (Garcia de Palazzo et al., Int J Biol Markers 1993, 8:233-239; Ross et al., Oncologist 2003, 8:307-325; Osman et al., J Urol 2005, 174:2174-2177; Kapitanovic et al., Gastroenterology 1997, 112:1103-1113; Turken et al., Neoplasma 2003, 50:257-261; and Oshima et al., Int J Biol Markers 2001, 16:250-254). The following table shows the expression status of HER2 in various tumor cell origins. (Table 1) Tumor cells and HER2 expression status JPEG0007644978000001.jpg38136 Trastuzumab is a recombinant humanized monoclonal antibody against HER2 protein domain IV, and thus blocks ligand-independent HER2 homodimerization and, to a lesser extent, heterodimerization of HER2 with other family members in cells in which HER2 is greatly overexpressed (Cho et al., Nature 2003, 421:756-760 and Wehrman et al., Proc Natl Acad Sci 2006, 103:19063-19068). Trastuzumab was found to inhibit the formation of HER2 / EGFR heterodimers in cells with moderate levels of HER2 expression (Wehrman et al., Proc Natl Acad Sci 2006, 103:19063-19068; Schmitz et al., Exp Cell Res 2009, 315:659-670). Trastuzumab mediates antibody-dependent cellular cytotoxicity (ADCC) and prevents ectodomain shedding, which would otherwise lead to the formation of a truncated, constitutively active protein in HER2-overexpressing cells. Trastuzumab has also been reported to inhibit the in vitro and in vivo growth of high levels of HER2-expressing tumor cells (reviewed in Nahta and Esteva, Oncogene 2007, 26:3637-3643). Trastuzumab has been approved by the NMPA and FDA for first-line and adjuvant treatment of HER2-overexpressing metastatic breast cancer in combination with chemotherapy or as a single agent following one or more chemotherapy regimens. Trastuzumab has been found to be effective in only 20-50% of patients with HER2-overexpressing breast cancer, with many initial responders relapsing after a few months (Dinh et al., Clin Adv Hematol Oncol 2007, 5:707-717).In the 2019 Breast Cancer Clinical Treatment Guidelines, the National Comprehensive Cancer Network (NCCN) states that pertuzumab and trastuzumab in combination with docetaxel (strength of recommendation 1) or paclitaxel as a first-line treatment regimen for HER2-positive late-stage (metastatic) breast cancer and new adjuvant therapy (i.e., before surgery) is expected to increase the success rate of breast cancer surgery, reduce the recurrence rate, and improve survival rate and quality of life. Pertuzumab (OmnitargTM) is another humanized monoclonal antibody. Pertuzumab is directed against HER2 protein domain II and is reported to inhibit ligand-induced heterodimerization (i.e., HER2 dimerization with another member of the ErbB family to which the ligand is bound), a mechanism that does not strictly require high HER2 expression levels (Franklin et al., Cancer Cell 2004, 5:317-328). Pertuzumab also mediates ADCC, but its primary mechanism of action is dependent on its inhibition of dimerization (Hughes et al., Mol Cancer THER 2009, 8:1885-1892). It has also been found that pertuzumab enhances EGFR internalization and downregulation by inhibiting the formation of EGFR / HER2 heterodimers, which would otherwise tether EGFR to the plasma membrane (Hughes et al., Mol Cancer THER 2009, 8:1885-1892). This is related to the observation that EGFR homodimers are internalized more efficiently than EGFR / HER2 dimers (Pedersen et al., Mol Cancer Res 2009, 7:275-284). According to the latest clinical study results of CLEOPATRA, NeoSphere, VELVET, and PERUSE, the combination of pertuzumab and trastuzumab, which target different epitopes of HER2, can significantly improve progression-free survival and median survival in patients with HER2-positive breast cancer. Another proposed HER2-based therapy is the combination of HER2 antibodies against different HER2 epitopes, which has been reported to be more effective than single HER2 antibodies in reducing tumor growth in in vitro and in vivo tumor models (Emde et al., Oncogene 2011, 30:1631-1642; Spiridon et al., Clin Cancer res 2002, 8:1720-1730). For example, the complementary mechanisms of action of pertuzumab and trastuzumab reportedly improve antitumor and antitumor efficacy when combined in patients who have progressed on prior trastuzumab therapy (Baselga et al., J Clin Oncol 2010, 28:1138-1144). A phase III clinical trial (CLEOPATRA) showed that the antibody combination of pertuzumab and trastuzumab, when used in combination with docetaxel, extended prolonged progression-free survival compared with the combination of trastuzumab and docetaxel in patients with previously untreated HER2-positive metastatic breast cancer. The combination of pertuzumab and trastuzumab has a more obvious effect than the single administration, and the adverse reaction rate of pertuzumab alone is only 3.4%, but the adverse reaction rate when combined with trastuzumab can reach 21.4%. Nevertheless, both trastuzumab and pertuzumab are only suitable for the treatment of breast cancer with strong HER2 positive (IHC test result 3+), and the therapeutic effect of both trastuzumab and pertuzumab on HER2 weakly positive breast cancer is not ideal. In recent years, cell therapy has achieved extraordinary results in the treatment of hematological tumors, so scientists are developing a treatment for HER2 positive breast cancer based on trastuzumab. As a result, the patient died of severe side effects on the fifth day after CAR-T cell infusion (Morgan et al., The American Society of Gene & Cell Therapy 2010, 4:843-851). Bispecific antibodies against different epitopes of HER2 are monoclonal antibody drugs for recognizing two different epitopes on the surface of HER2, which contain two different antibody heavy chain variable regions and two identical light chain variable regions (CN105829347A, CN105820251A, CN105980409A), and can specifically bind to the second and fourth domains on the surface of HER2, with the aim of providing a bispecific antibody with trastuzumab activity and pertuzumab activity, to replace the current clinical treatment using two kinds of antibodies, trastuzumab and pertuzumab in combination. However, the antibodies in the prior art (CN105829347A, CN105820251A, CN105980409A), have the characteristics of low binding activity to antigens or low stability, and are prone to precipitation, and stability is a prerequisite for antibody drug discovery. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the above technical deficiencies of the prior art, such as low affinity, low stability, and easy precipitation, the present invention aims to provide a bispecific antibody against different epitopes of HER2 and its application. The inventors of the present application have discovered that by using the heavy chains of pertuzumab and trastuzumab, respectively, and optimizing the natural trastuzumab light chain as a common light chain, a bispecific monoclonal antibody can be obtained whose affinity for the HER2 target second and fourth domains is equivalent to that of wild-type pertuzumab and trastuzumab, respectively. Due to the characteristics of bispecificity and high affinity, the antibody of the present invention is likely to be effective against HER2 weakly positive breast cancer. At the same time, it has been unexpectedly discovered that the antibody of the present invention has an enhanced growth inhibitory effect on various HER2-expressing tumor cells. [Means for solving the problem]

[0005] In order to solve the above technical problems, the technical means of the first aspect of the present invention is as follows: A bispecific antibody against HER2 is provided, comprising a first protein domain for recognizing the extracellular domain IV and the extracellular domain II of HER2 and a second protein domain, the first protein domain comprises a first heavy chain and a light chain, the second protein domain comprises a second heavy chain and a light chain, the first heavy chain comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO:4 or a mutant amino acid sequence thereof, the second heavy chain comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO:3 or a mutant amino acid sequence thereof, the light chain comprises a light chain variable region, and the light chain variable region has an amino acid substitution at one or more sites selected from R24, N30, T31, A32, F53, L54, S56, R66, H91, T93, T94 and P96 in the amino acid sequence shown in SEQ ID NO:1. The bispecific antibody may be an scFv, F(ab)2 or IgG-like structure. In a preferred specific embodiment, the bispecific antibody comprises an amino acid substitution in the light chain variable region, where the original amino acid residue is replaced with an amino acid residue selected from Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Gly (G), Ala (A), Val (V), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Tyr (Y), Trp (W) and Arg (R). In a preferred specific embodiment, the bispecific antibody has one or more amino acid residue substitutions selected from R24K, N30S, T31I, A32G, F53Y, L54R, S56T, R66G, H91Y, T93I, T94Y and P96Y in the amino acid sequence shown in SEQ ID NO: 1 in the light chain variable region, preferably at one or more sites selected from N30S, F53Y, L54R, S56T and P96Y. In a preferred specific embodiment, the bispecific antibody has, in the light chain variable region, an amino acid substitution in the amino acid sequence shown in SEQ ID NO:1 selected from the group consisting of: (1) F53Y; (2) F53Y, L54R and S56T; (3) P96Y; (4) N30S and F53Y; (5) N30S, F53Y, L54R and S56T; or (6) N30S, P96Y. In a preferred specific embodiment, the bispecific antibody further comprises a light chain constant region, which is preferably a human light chain constant region λ light chain or κ light chain. Preferably, the light chain has amino acid residue substitutions in the amino acid sequence shown in SEQ ID NO:2 selected from the group consisting of: (1) F53Y, (2) F53Y, L54R and S56T, (3) P96Y, (4) N30S and F53Y, (5) N30S, F53Y, L54R and S56T, or (6) N30S, P96Y. In a preferred specific embodiment, the first and second heavy chains of the bispecific antibody further comprise a heavy chain constant region, which is preferably a human heavy chain constant region. Preferably, the first and second heavy chains of the bispecific antibody form a heterodimer, and the first and second heavy chains are preferably connected by a knob-in-hole structure. More preferably, the heavy chain constant region of the second heavy chain has a D102E mutation in the amino acid sequence shown in SEQ ID NO:3. In a preferred specific embodiment, the heavy chain constant region of the first heavy chain of the bispecific antibody comprises the CDR sequences of HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NOs:6-13, and HCDR3 as set forth in SEQ ID NOs:14-19, Preferably, the heavy chain constant region of the first heavy chain is selected from the group consisting of HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:6, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:16, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:9, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:17, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:17, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:15, HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:17 ... HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:11, HCDR3 shown in SEQ ID NO:17, or HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:12, HCDR3 shown in SEQ ID NO:17, or HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:17, or HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:18, or HCDR1 shown in SEQ ID NO:5, or HCDR2 shown in SEQ ID NO:7, and HCDR3 shown in SEQ ID NO:19. In a preferred specific embodiment, said bispecific antibody has reduced fucose modification, preferably said antibody is a non-fucose modified antibody. In order to solve the above technical problems, the technical means of the second aspect of the present invention are as follows: A base sequence encoding the bispecific antibody or the antigen-binding portion thereof described above is provided. In order to solve the above technical problems, the technical means of the third aspect of the present invention are as follows: An expression vector containing the base sequence of the second aspect is provided. In order to solve the above technical problems, the technical means of the fourth aspect of the present invention are as follows: A prokaryotic or eukaryotic cell comprising the expression vector of the third aspect is provided. In order to solve the above technical problems, the technical means of the fifth aspect of the present invention is as follows: A light chain is provided, which comprises a light chain variable region, and in the light chain variable region, an amino acid residue substitution selected from the group consisting of (1) F53Y, (2) F53Y, L54R and S56T, (3) P96Y, (4) N30S and F53Y, (5) N30S, F53Y, L54R and S56T, or (6) N30S, P96Y is performed in the amino acid sequence shown in SEQ ID NO: 1. Preferably, the light chain further comprises a light chain constant region, and the light chain constant region is preferably a human light chain constant region λ light chain or κ light chain. More preferably, the light chain has amino acid residue substitutions in the amino acid sequence set forth in SEQ ID NO:2 selected from the group consisting of: (1) F53Y; (2) F53Y, L54R and S56T; (3) P96Y; (4) N30S and F53Y; (5) N30S, F53Y, L54R and S56T; or (6) N30S, P96Y. In order to solve the above technical problems, the technical means of the sixth aspect of the present invention is as follows: providing an application of the light chain in the above fifth aspect in preparing an antibody, preferably, the antibody is an scFv, Fab', F(ab)2 or full-length antibody, or a monoclonal antibody, a bispecific antibody or a multispecific antibody; In order to solve the above technical problems, the technical means of the seventh aspect of the present invention is as follows. A bispecific antibody-drug conjugate against HER2 is provided, in which the bispecific antibody described above is covalently bound to a cytotoxic drug. Preferably, the cytotoxic drug is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA minor groove inhibitor. More preferably, the tubulin inhibitor is a maytansinoid compound, or an auristatin derivative, or tubulin, the topoisomerase inhibitor is DXd or a derivative thereof, or SN-38, or adriamycin and a derivative thereof, and the DNA minor groove inhibitor is calicheamicin, duocarmycin and a derivative thereof, pyrrolobenzodiazepine compounds, or indolinobenzodiazepine compounds. In order to solve the above technical problems, the technical means of the eighth aspect of the present invention are as follows: A pharmaceutical composition comprising the bispecific antibody described above is provided. Preferably, the pharmaceutical composition further comprises hyaluronidase. More preferably, the hyaluronidase is rHuPH20 or modified rHuPH20. Most preferably, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or excipient. In order to solve the above technical problems, the technical means of the ninth aspect of the present invention are as follows. (1) constructing a nucleotide sequence encoding the heavy chain and a nucleotide sequence encoding the light chain in the same vector or in different vectors; and (2) expressing the vector constructed in the above step in a different host cell or in the same host cell. In order to solve the above technical problems, the technical means of the tenth aspect of the present invention is as follows: providing the use of the above-mentioned bispecific antibody against HER2, the bispecific antibody-drug conjugate against HER2, and the pharmaceutical composition comprising the bispecific antibody in the preparation of a medicine for treating the above-mentioned HER2-expressing tumors, preferably, the positive tumors include breast cancer, gastric cancer, prostate cancer, lung cancer, bladder cancer, ovarian cancer, colon cancer, esophageal cancer, head and neck cancer, endometrial cancer, malignant melanoma, pharyngeal cancer, oral cancer, and skin cancer, and more preferably, breast cancer, gastric cancer, and lung cancer; In order to solve the above technical problems, the technical means of the eleventh aspect of the present invention is as follows: A kit combination is provided, comprising Kit A containing the above-described bispecific antibody against HER2, bispecific antibody-drug conjugate against HER2, and pharmaceutical composition containing the bispecific antibody, and Kit B containing another antibody, bispecific antibody, recombinant cell, or pharmaceutical composition targeting HER2 or another target. The kits A and B are used in random order, and Kit A is used before Kit B, or Kit B is used before Kit A. The bispecific antibody against HER2, the immunoconjugate and the pharmaceutical composition or the kit combination of the present invention can be administered to a patient to treat the associated tumor. In the present invention, the HER2-recognizing antibody of the present invention or its antigen-binding portion or a mixture thereof, or therapeutic immune cells containing the antigen-binding portion thereof can also be used in combination with a chemotherapeutic drug and / or other antibodies, and therefore the composition of the present invention can further contain a chemotherapeutic drug and / or other antibodies. In the present invention, the chemotherapeutic agents include, but are not limited to, adriamycin, cyclophosphamide and taxanes [paclitaxel (Taxol) and docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (anastrozole, Femara tablets, Aromasin tablets), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramusting, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), etc., or combinations thereof. In accordance with common knowledge in this field, the above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention. All reagents and materials used in the present invention are commercially available. Effect of the Invention

[0006] The positive and progressive effect of the present invention is to obtain a bispecific monoclonal antibody with high affinity, excellent stability, strong tumor cell killing activity, and high ADCC activity by mutating specified amino acids in the light chain variable region of trastuzumab, the heavy chain variable region of pertuzumab, and the heavy chain variable region of trastuzumab. [Brief description of the drawings]

[0007] [Figure 1] 1 shows the results of the growth inhibitory activity against MDA-MB-175 cells of bispecific antibodies against different epitopes of human HER2. A shows a comparison between KJ015w and pertuzumab, and B shows a comparison between trastuzumab and pertuzumab. [Diagram 2] FIG. 1 shows protein electrophoresis diagrams of bispecific monoclonal antibodies. [Diagram 3] 1 shows the cell binding activity of bispecific antibodies against different epitopes of human HER2. [Figure 4]1 shows the results of endocytosis of bispecific antibodies against different epitopes of human HER2 in NCI-N87 (A) and MCF-7 (B) cells. [Diagram 5] This shows the results of the growth inhibitory activity of bispecific antibodies against different epitopes of human HER2 against MDA-MB-175 cells. A is the result for KJ015-A, B is the result for KJ015-B, C is the result for KJ015-C, and D is the result for KJ015-D. [Figure 6] This shows the results of the growth inhibitory activity against BT474 cells of bispecific antibodies against different epitopes of human HER2. A is the result for KJ015-A, B is the result for KJ015-B and KJ015-C, and C is the result for KJ015-D. [Figure 7] This shows the results of the growth inhibitory activity against SK-BR-3 cells of bispecific antibodies against different epitopes of human HER2. A is the result for KJ015-A, B is the result for KJ015-B and KJ015-C, and C is the result for KJ015-D. [Figure 8] 1 shows the results of the growth inhibitory activity against NCI-N87 cells of bispecific antibodies against different epitopes of human HER2. A shows the results for KJ015-A and KJ015-B, and B shows the results for KJ015-C and KJ015-D. [Figure 9] This shows the results of the growth inhibitory activity against Calu-3 cells of bispecific antibodies against different epitopes of human HER2. A shows the results for KJ015-A and KJ015-C, B shows the results for KJ015-B, and C shows the results for KJ015-D. [Figure 10] The results of ADCC activity of bispecific antibodies against different epitopes of human HER2 against JIMT-1 cells are shown in Fig. 1. A shows the results between KJ015-A and KJ015-B, and B shows the results between KJ015-C and KJ015-D. [Figure 11] 1 shows the results of ADCC activity against SK-BR-3 cells of bispecific antibodies against different epitopes of human HER2. A shows the results between KJ015-A and KJ015-B, and B shows the results between KJ015-C and KJ015-D. [Figure 12] The results of ADCC activity of bispecific antibodies against different epitopes of human HER2 against NCI-N87 cells are shown. A shows the results for KJ015-A and KJ015-B, B shows the results for KJ015-C and KJ015-D, and C shows the results for KJ015-E. [Figure 13] 1 shows the results of ADCC activity against MDA-MB-175 cells of bispecific antibodies against different epitopes of human HER2; A shows the results for KJ015-A to KJ015-C, and B shows the results for KJ015-D and KJ015-E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The technical means of the present invention are further described below through specific embodiments. Those skilled in the art should understand that the examples are only for understanding the present invention, and should not be regarded as specifically limiting the present invention. In the following examples, experimental methods for which no specific conditions are specified are carried out according to conventional methods and conditions or product manuals. Example 1 Construction and expression of bispecific antibodies against distinct epitopes of human HER2. DNA fragments encoding the trastuzumab heavy chain (including the knob structure), the pertuzumab heavy chain (including the hole structure) and the trastuzumab light chain (SEQ ID NO: 2) were synthesized by total gene synthesis and cloned into the constructed expression vectors. 1) the glutamine synthetase gene as a selection marker, or the neomycin resistance gene as a heavy chain selection marker; 2) an origin of replication, which is the ori; 3) an origin of replication from the vector pUC18 that allows this plasmid to replicate in E. coli; 4) the β-lactamase gene that confers ampicillin resistance to E. coli; 5) the immediate early enhancer and promoter from human cytomegalovirus; 6) human 1 - composed of the immunoglobulin polyadenylation ("poly A") signal sequence and As described above, immunoglobulin genes containing heavy or light chains are prepared by gene synthesis and cloned into pUC57(Amp) plasmid. Using HindIII and EcoRI, pUC57(Amp) plasmid containing heavy chain gene, pUC57(Amp) plasmid containing light chain gene, and corresponding expression plasmids are enzymatically cleaved, and heavy and light chains are directionally cloned into the corresponding plasmids, respectively. The enzymatic cleavage and ligation procedures are carried out according to the instructions of the commercially available kit. The above constructed trastuzumab heavy chain, pertuzumab heavy chain and light chain expression vectors are transformed into E. coli DH5α, and positive clones are taken out and inoculated into 500 ml of LB medium and grown. DNA is extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit according to the manufacturer's instructions. Plasmid DNA containing the above heavy and light chain coding sequences is co-introduced into CHO-K1 (Chinese hamster ovary cells purchased from ATCC) at a ratio of 1:1:2 to 1:1:4 using Invitrogen's lipofection kit, and the operation is performed according to the manufacturer's instructions. 24-48 hours after the introduction, the cell medium is changed to a selective medium containing a selective drug, and the selective medium is changed every 3-4 days until cell clones are formed. When the diameter of the cell clones is 1-2 mm, a monoclone is removed from the plate using a cloning ring and transferred to a 24-well plate. When the single cell clones grow to fill 50-70% of the well plate in the 24-well plate, the culture supernatant of each clone is taken and subjected to ELISA detection, and cells with high expression levels and good growth conditions are selected and cultured for expansion. For the above ELISA detection method, trastuzumab-specific antigen (Rhinogen®) and pertuzumab-specific antigen (Rhinogen®) are each diluted to a specific concentration (2 μg / ml) with coating solution (pH 9.6 CBS) and coated on a 96-well plate at 100 μl / well and left at 2-8 ° C overnight. Discard the liquid in the well, wash three times with PBST, and after dehydration, add 300 μl / well of blocking solution (1% BSA PBST), leave at room temperature for 2 hr, wash three times with PBST, and dehydrate. Standard trastuzumab and pertuzumab are diluted with diluent PBS (pH 7.0) and diluted 2-fold from 100 ng / mL to obtain seven gradient concentration samples. Dilute the expression supernatant appropriately according to the situation, apply it to a 96-well plate with duplicate wells at 100 μl / well, incubate at 37 °C for 1 hr, discard the solution, wash the plate three times, and dehydrate. Dilute the enzyme-linked antibody (HRP-light chain specific antibody against human IgGγ, Thermo) to a specific concentration (1:2000) with diluent, add it to a 96-well plate with 100 μl / well, react at 37 °C for 1 hr, discard the solution, wash the plate three to six times, and dehydrate. Prepare a substrate mixture, add it to a 96-well plate with 100 μl / well, and incubate at 37 °C for 20 min. Add the reaction termination solution at 50 μl / well to stop the reaction. Read the absorbance value OD at 490 nm and calculate the content of the sample according to the standard curve. Finally, wild-type KJ015w antibody expressing cell lines are obtained. Each cell line is grown in CD2 basal medium (Rhinogen®) to a volume of 100 ml, fed-batch cultured, and supplemented with 2% Feed4 (Rhinogen®) every day from day 3. After 12 days of suspension culture, the culture supernatants are collected and purified by Protein A affinity chromatography to obtain bispecific monoclonal antibody protein samples. Example 2 Affinity measurement of bispecific antibodies against different epitopes of human HER2 and HER2 Using Biacore T200, we will measure the affinity of the single-targeting HER2 antibodies trastuzumab and pertuzumab, and the HER2 bispecific antibody KJ015w, to three types of HER2 antigens. First, the anti-human Fc (AHC) antibody was bound to a CM5 chip, and then the target antibody IgG (2 μg / ml) was captured by the AHC. Three types of antigen molecules at different concentrations were allowed to flow over the chip surface that captured the IgG, and the binding activity between the HER2 antibody and the antigens at different concentrations was detected. The obtained data was fitted using Biacore T200 analysis software to obtain accurate kinetic constants, and the comparison results are shown in Table 2. (Table 2) Measurement results of affinity between antibody KJ015w and three types of HER2 antigen JPEG0007644978000002.jpg92136 Trastuzumab, pertuzumab and bispecific antibody KJ015w against HER2 can all bind to the full-length HER2 extramembrane domain antigen, and bispecific antibody KJ015w against HER2 can recognize not only pertuzumab HER2-specific antigen (Rhinogen®) but also trastuzumab HER2-specific antigen (Rhinogen®). However, the affinity of pertuzumab to HER2-specific antigen is two orders of magnitude lower than that of pertuzumab. Example 3 Detection of cell proliferation inhibitory activity of bispecific antibodies against different epitopes of human HER2 Place the MDA-MB-175 cell culture flask in a 5% carbon dioxide incubator at 37°C for static culture, observe the color of the medium and cell confluency daily, and record the cell doubling time. Subculture the cells every 2-4 days. When the cells fill approximately 80% of the flask, digest and inoculate onto plates. Discard the medium, wash once with PBS, and add 0.25% trypsin (Gibco) for digestion. Pipette the cells into a centrifuge tube and centrifuge at 500g for 3 min. Discard the supernatant, add complete medium to resuspend the cells, and take 100 μL of the cell suspension for counting. Adjust the cell density to 1 × 10 in 2% FBS-containing medium. 5Adjust the concentration to cells / mL and inoculate into a 96-well plate at 100 μL per well. Dilute the drug to be measured with 2% FBS-containing medium. First, dilute the drug to be measured to 150 μg / mL, then dilute it 8 times with a 4-fold gradient to obtain 9 concentration gradients of the drug, and the last point is the negative control point. The antibody initial concentration varies depending on the cells, and for details, refer to the initial concentration points in the result diagram. Add 100 μL of the diluted drug to the corresponding wells of the 96-well plate inoculated with the cells, so that there are two duplicate wells for each point. Place the 96-well plate in a carbon dioxide incubator and incubate and react for 96 to 120 hours. After the incubation is completed, add CCK8 reagent (Rhinogen®) at 20 μL / well to the 96-well plate and shake on a microplate shaker to mix evenly. Read the absorbance values ​​at OD 450 nm with a microplate reader 0.5-6 h after staining. Perform curve fitting analysis on the raw data using GraphPad software. The results of the inhibitory activity of the antibody against proliferation (FIG. 1A) show that KJ015w has proliferation inhibitory activity against MDA-MB-175 cells. Example 4 Construction and expression of bispecific antibodies against distinct epitopes of human HER2. Three types of DNA fragments encoding the light chain mutants are synthesized by total gene synthesis [the amino acid sequence shown in SEQ ID NO:2 is substituted with amino acid residues selected from the group consisting of F53Y (M1), F53Y, L54R, S56T (M2), and P96Y (M3)], and cloned into the heavy and light chain expression vectors of the constructed antibody, respectively. The above constructed trastuzumab heavy chain, pertuzumab heavy chain and light chain expression vectors are transformed into E. coli DH5α respectively, and positive clones are taken out and inoculated into 500 ml of LB medium and grown. DNA is extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit according to the manufacturer's instructions. Plasmid DNA containing the above heavy chain and light chain coding sequences is co-introduced into CHO-K1 at a specific ratio (1:1:2) using Invitrogen's lipofection kit, and the operation is performed according to the manufacturer's instructions. 24-48 hours after the introduction, the cell medium is changed to a selective medium containing a selective drug, and the selective medium is changed every 3-4 days until cell clones are formed. When the diameter of the cell clones is 1-2 mm, a monoclone is removed from the plate using a cloning ring and transferred to a 24-well plate. When the single cell clones grow to fill 50-70% of the well plate in the 24-well plate, the culture supernatant of each clone is taken and subjected to ELISA detection, and cells with high expression levels and good growth conditions are selected and cultured for expansion. For the above ELISA detection method, trastuzumab-specific antigen (Rhinogen®) and pertuzumab-specific antigen (Rhinogen®) are each diluted to a specific concentration (2 μg / ml) with coating solution (pH 9.6 CBS) and coated on a 96-well plate at 100 μl / well and left at 2-8 ° C overnight. Discard the liquid in the well, wash three times with PBST, and after dehydration, add 300 μl / well of blocking solution (1% BSA PBST), leave at room temperature for 2 hr, wash three times with PBST, and dehydrate. Standard trastuzumab and pertuzumab are diluted with diluent PBS (pH 7.0) and diluted 2-fold from 100 ng / mL to obtain seven gradient concentration samples. Dilute the expression supernatant appropriately according to the situation, apply it to a 96-well plate with duplicate wells at 100 μl / well, incubate at 37 °C for 1 hr, discard the solution, wash the plate three times, and dehydrate. Dilute the enzyme-linked antibody (HRP-light chain specific antibody against human IgGγ, Thermo) to a specific concentration (1:2000) with diluent, add it to a 96-well plate with 100 μl / well, react at 37 °C for 1 hr, discard the solution, wash the plate three to six times, and dehydrate. Prepare a substrate mixture, add it to a 96-well plate with 100 μl / well, and incubate at 37 °C for 20 min. Add the reaction termination solution at 50 μl / well to stop the reaction. Read the absorbance value OD at 490 nm and calculate the content of the sample according to the standard curve. Finally, wild-type mutant KJ015m1, KJ015m2 and KJ015m3 antibody-expressing cell lines are obtained. Each cell line is grown in CD2 basal medium (Rhinogen®) to a volume of 100 ml, fed-batch cultured, and supplemented with 2% Feed4 (Rhinogen®) every day from the third day. After 12 days of suspension culture, the culture supernatants are collected and pre-purified by Protein A affinity chromatography to obtain three bispecific monoclonal antibody protein samples with a common light chain (Figure 2). Figure 2 shows that the protein purity of the three bispecific monoclonal antibodies is high. Example 5 Affinity detection of bispecific antibodies against different epitopes of human HER2 and HER2 Trastuzumab-specific antigen (Rhinogen®), pertuzumab-specific antigen (Rhinogen®), and HER2 full-length antigen (Rhinogen®) are each diluted to a specific concentration (2 μg / ml) in coating solution (pH 9.6 CBS) and coated on a 96-well plate at 100 μl / well and left overnight at 2-8 ° C. Discard the liquid in the well, wash three times with PBST, and after dehydration, add 300 μl / well of blocking solution (1% BSA PBST), leave at room temperature for 2 hr, wash three times with PBST, and dehydrate. Dilute the standard trastuzumab and pertuzumab with diluent PBS (pH 7.0), dilute 3-fold from 2000ng / mL to obtain 10 gradient concentration samples, apply 100μl / well of duplicate wells to a 96-well plate, incubate at 37℃ for 1hr, discard the solution, wash the plate 3 times, and dehydrate. Dilute enzyme-linked antibody (HRP-light chain specific antibody against human IgGγ, Thermo) to a specific concentration (1:2000) with diluent, add 100μl / well to a 96-well plate, react at 37℃ for 1hr, discard the solution, wash the plate 3-6 times, and dehydrate. Prepare a substrate mixture, add 100μl / well to a 96-well plate, and incubate at 37℃ for 20min. Add 50μl / well of reaction termination solution to stop the reaction. (Table 3) ELISA affinity detection results between each HER2 antibody and antigens for different epitopes of HER2 JPEG0007644978000003.jpg55136Note: **P<0.01 compared to KJ015w, ***P<0.001 compared to KJ015w. The results in Table 3 show that the binding affinity of the bispecific antibodies KJ015m1, KJ015m2, and KJ015m3 of the present invention to the Pertuzumab-specific antigen is higher than that of the wild type (i.e., KJ015w) (P<0.001). The binding of the bispecific antibodies of the present invention to the Trastuzumab-specific antigen is equivalent to that of the wild type, and the affinity of KJ015m3 is slightly superior to that of the wild type (P<0.01). In the binding test to the HER2 full-length antigen, the affinity of KJ015m1 and KJ015m2 is equivalent to that of the wild type, and the affinity of KJ015m3 is clearly superior to that of the wild type (P<0.001). Example 6 Thermal stability detection of bispecific antibodies against different epitopes of human HER2 The antibody to be detected is diluted to 1mg / ml in PBS, and SYPRO orange dye (Life technologies) is diluted to 40x in deionized water. Take 12.5μL of the diluted sample, add 2.5μl of 40x dye, 5μL of deionized water, and load into duplicate wells at 20μl per well. After sealing with sealing film, place in a PCR machine and react with a PCR reaction program of 25℃ for 2 minutes and 95℃ for 2 minutes. The original reading value is derived, and the results are introduced into GraphpadPrism to perform curve fitting analysis. The Tm value of each detected sample is shown in Table 4, and the results show that the thermal stability of the M1 to M3 light chain mutants is all good. (Table 4) Thermal stability data of bispecific antibodies against different epitopes of human HER2 JPEG0007644978000004.jpg29136 (Example 7) Heavy chain modification of bispecific antibodies against different epitopes of human HER2 The light chain variants M1-3 are selected as light chains to perform heavy chain mutation modification of Pertuzumab. Using the mixed light chains of M1, M2, and M3, high-throughput mutation is performed on the target region of a single heavy chain template using Kunkel technology to construct two libraries: Library 1, in which the mutation region is in HCDR1, HCDR2, and HCDR3, and Library 2, in which the mutation region is in HCDR2. The supercompetent cells are electrically transformed and cultured to obtain phage library 1 and library 2, respectively. Using antigen recombinant protein HER2ECD (Rhinogen®) as bait, titer test is performed to confirm whether phages are enriched or not based on solid-phase screening in maxi-sorp 96-well plate. Screening is performed using library 1 and library 2. For each enriched phage of each library, 48 monoclones are selected and cultured. The cultured phages are collected and subjected to ELISA identification. Positive monoclones with higher affinity than wild-type antibody are selected and subjected to DNA sequencing to obtain antibody sequences. Example 8 Finding affinity constants for candidate sequences Based on the plasmid of Example 1, the plasmid DNA of the coding sequence of the heavy chain and the light chain is co-transfected into ExpiCHO-S cell line (Gibco) at a specific ratio (1:1), and the operation is performed according to the instructions of the Rhinogen (registered trademark) transfection kit. After the transfection, the cells are cultured for 7 to 10 days, and the culture supernatant is taken and purified by Protein A affinity chromatography for detection of the affinity constant. The affinity between the single target HER2 antibody and the HER2ECD antigen (Rhinogen®) is measured using Biacore T200. First, the Anti-Human Fc (AHC) antibody is bound to a CM5 chip, and the antibody IgG (2 μg / ml) to be measured is captured by AHC. Three types of antigen molecules with different concentrations are allowed to flow over the chip surface where the IgG is captured, and the binding activity between the HER2 antibody and the antigen with different concentrations is detected, and the obtained data is fitted by Biacore T200 analysis software to obtain accurate kinetic constants. The detailed affinity constant results are shown in Table 5. According to the affinity constant data, the affinity constant of the affinity-modified pertuzumab heavy chain is increased by two orders of magnitude compared to the wild-type pertuzumab heavy chain. The D102E trastuzumab heavy chain has an affinity improved by about three times compared to the wild-type trastuzumab heavy chain. (Table 5) Affinity constant of the candidate sequence JPEG0007644978000005.jpg220136JPEG0007644978000006.jpg109136Note: The candidate heavy chains in Table 5 only have mutations in the HCDR2 and HCDR3 regions compared to the variable region sequences of the heavy chain of Pertuzumab, and the HCDR1 sequences are all SEQ ID NO:5. Example 9 Preparation of bispecific antibodies against different epitopes of human HER2 Based on the plasmid of Example 1, gene synthesis of antibody heavy chain is performed according to Table 7, and the corresponding heavy chain plasmid is obtained by plasmid extraction. The same method is used to construct N30S M1 light chain, N30S M2 light chain and D102E trastuzumab heavy chain. Using Rhinogen (registered trademark) transient transfection kit, plasmid DNA containing the above heavy chain and light chain coding sequences is co-transfected into ExpiCHO-S cell line (Gibco) at a specific ratio (1:1:2), and the operation is performed according to the instructions of the Rhinogen (registered trademark) transfection kit. After transfection, the cells are cultured for 7 to 10 days, and the culture supernatant is taken and purified by protein A affinity chromatography for functional evaluation. After purification, the sample is sterilized and filtered using a membrane filter with a pore size of 0.22 μm and stored at 2 to 8 ° C. The obtained sample information is shown in Table 6. During the storage of the samples, only the KJ015-E sample exhibited a precipitation phenomenon, while the other samples were transparent, indicating that the KJ015-E sample was unstable. (Table 6) HER2 Bispecific Antibody Sequence Combinations JPEG0007644978000007.jpg51136#: The heavy chain sequence number is based on the SEQ ID NO of patent US20170029529. Example 10 Purity detection of bispecific antibodies against different epitopes of human HER2 Purity detection of KJ015 antibody is performed by size exclusion chromatography (Chinese Pharmacopoeia 2010, Part 3, Appendix IIIB). Hydrophilic modified silica gel is used as the packing material (TSKgel SuperSW mAb HR, 300×7.8 mm), and a mixed solution of disodium hydrogen phosphate and isopropanol (28.4 g of disodium hydrogen phosphate is taken, dissolved by adding purified water, diluted to 800 ml, pH adjusted to 7.0 with phosphoric acid, 10 ml of isopropanol is added, and the volume is adjusted to 1 L with purified water) is used as the mobile phase, the flow rate is 0.7 ml per minute, and the detection wavelength is 280 nm. 20 μl of the sample solution is taken and injected into the liquid chromatography for detection. The peak area of ​​the antibody monomer is calculated by the peak area normalization method. CE-SDS detection of KJ015 sample is performed using a PA800plus instrument, referring to the USP Medicines Compendium Bevacizumab and Beckman Coulter official documents. The purity values ​​of each detected sample are shown in Table 7. The purity of each antibody was high and close to that of trastuzumab and pertuzumab, indicating that they could be used for subsequent activity evaluation. (Table 7) Purity of each antibody sample to be detected JPEG0007644978000008.jpg43136 (Example 11) Thermal stability detection of bispecific antibodies against different epitopes of human HER2 The antibody to be detected is diluted to 1mg / mL in PBS, and SYPRO orange dye (Life technologies) is diluted to 40x in deionized water. Take 12.5μL of the diluted sample, add 2.5μl of 40x dye, 5μL of deionized water, and load into duplicate wells at 20μl per well. After sealing with sealing film, place in a PCR machine and react with a PCR reaction program of 25℃ for 2 minutes and 95℃ for 2 minutes. The original reading value is derived, and the results are introduced into GraphpadPrism for curve fitting analysis. The Tm value of each detected sample is shown in Table 8. It can be seen that there is little difference in the Tm value of each antibody, which is close to trastuzumab and pertuzumab. (Table 8) Tm value of each sample JPEG0007644978000009.jpg38137 (Example 12) Cell-binding activity of bispecific antibodies against different epitopes of human HER2 Place the cell culture flask in a 37°C, 5% carbon dioxide incubator for static culture, observe the color of the medium and cell confluency daily, and record the cell doubling time. Subculture the cells every 2 to 4 days. When the cells fill approximately 80% of the flask, digest and inoculate onto plates. Discard the medium, wash once with PBS, and add 0.25% trypsin (Gibco) for digestion. Pipette the cells into a centrifuge tube and centrifuge at 500 g for 3 min. Discard the supernatant, add complete medium to resuspend the cells, and take 100 μL of the cell suspension for counting. Resuspend the cells in PBS (containing 1% BSA) and adjust the cell density to 3 × 10 6 Adjust to cells / mL. Dilute the drug to be measured with 2% FBS-containing medium. First, dilute the drug to be measured to a certain concentration, then dilute it five times with a 3-fold gradient to obtain six concentration gradients of the drug. The initial antibody concentration varies depending on the cells, and for details, refer to the initial concentration point in the result diagram. Take 100 μL of resuspended cells, add the antibody to be detected with a final concentration of 300 nM to 1 nM, and incubate at 4 ° C for 1 hour with isotype IgG (Rhinogen (registered trademark)) as the negative control. After the incubation is completed, wash three times with pre-cooled PBS, resuspend the cells in 50 μL of PBS (containing 1% BSA), add anti-human fluorescent secondary antibody, and incubate at 4 ° C for half an hour. After the incubation is completed, wash three times with pre-cooled PBS, and place in the detection device for detection. The detection results (FIG. 3) show that all of the KJ015A to D samples had binding activity, which was higher than that of trastuzumab and was equivalent to the results of the mixed sample of trastuzumab + pertuzumab. Example 13 Endocytic activity of bispecific antibodies against different epitopes of human HER2. Place the cell culture flask in a 37°C, 5% carbon dioxide incubator for static culture, observe the color of the medium and cell confluency daily, and record the cell doubling time. Subculture the cells every 2 to 4 days. When the cells fill approximately 80% of the flask, digest and inoculate onto plates. Discard the medium, wash once with PBS, and add 0.25% trypsin (Gibco) for digestion. Pipette the cells into a centrifuge tube and centrifuge at 500 g for 3 min. Discard the supernatant, add complete medium to resuspend the cells, and take 100 μL of the cell suspension for counting. Resuspend the cells in PBS (containing 1% BSA) and adjust the cell density to 3 × 10 6 Adjust to cells / mL. Take 100 μL of resuspended cells, add the antibody to be detected at a final concentration of 10 μg / mL (NCI-N87) or 50 μg / mL (MCF-7), and use isotype IgG (Rhinogen®) as a negative control, and incubate at 4°C for 1 hour. Wash three times with pre-chilled PBS, resuspend the cells in 100 μL of complete medium, and incubate in a cell incubator at 37°C for 1 hour. After incubation is complete, wash three times with pre-chilled PBS, resuspend the cells in 50 μL of PBS (containing 1% BSA), add anti-human fluorescent secondary antibody, and incubate at 4°C for half an hour. After incubation is complete, wash three times with pre-chilled PBS, and place in a detection device for detection. The endocytosis rate of each antibody to be detected at each time point was calculated and plotted according to the following formula: Endocytosis (%) = 1 - (MFI of sample-treated group at a certain time point - MFI of control hIgG at that time point) / (MFI of sample-treated group at 0 hours - MFI of control hIgG at 0 hours) x 100%. The endocytosis results are shown in Figure 4. In NCI-N87 cells, the endocytosis efficiency of all four samples was between that of trastuzumab and trastuzumab + pertuzumab. In MCF-7 cells, the activity of the KJ015-A sample was slightly lower than that of trastuzumab and trastuzumab + pertuzumab, the activity of the KJ015-B sample was equivalent to that of these two antibodies, and the activity of the KJ015-C and KJ015-D samples was higher than that of trastuzumab and the combination of trastuzumab + pertuzumab. Example 14 Growth inhibitory activity of bispecific antibodies against different epitopes of human HER2. The cell proliferation inhibition method was used to evaluate the ability of antibodies against different mutants of HER2 to inhibit the expression of HER2-positive tumor cells, and the cell lines used are shown in Table 9. Place the cell culture flask in a 37°C, 5% carbon dioxide incubator for static culture, observe the color of the medium and cell confluency daily, and record the cell doubling time. Subculture the cells every 2 to 4 days. When the cells fill approximately 80% of the flask, digest and inoculate onto plates. Discard the medium, wash once with PBS, and add 0.25% trypsin (Gibco) for digestion. Pipette the cells into a centrifuge tube and centrifuge at 500 g for 3 min. Discard the supernatant, add complete medium to resuspend the cells, and take 100 μL of the cell suspension for counting. Adjust the cell density to 1 × 10 in 2% FBS-containing medium. 5 Adjust the concentration to cells / mL and inoculate into a 96-well plate at 100 μL per well. Dilute the drug to be measured with 2% FBS-containing medium. First, dilute the drug to be measured to 20-6000μg / mL, then dilute 8-10 times with a gradient of 2.2-3 times to obtain 9-11 concentration gradients of the drug, and the last point is the negative control point. The antibody initial concentration varies depending on the cells, and for details, refer to the initial concentration points in the result diagram. Add 100μL of the diluted drug to the corresponding wells of the 96-well plate inoculated with the cells, so that there are two duplicate wells for each point. Place the 96-well plate in a carbon dioxide incubator and incubate and react for 96-120 hours. After the incubation is completed, add CCK8 reagent (Rhinogen®) at 20 μL / well to the 96-well plate and shake on a microplate shaker to mix evenly. Read the absorbance values ​​at OD 450 nm with a microplate reader 0.5-6 h after staining. Perform curve fitting analysis on the raw data using GraphPad software. (Table 9) Cell proliferation inhibition rate JPEG0007644978000010.jpg87136The results of MDA-MB-175 cell proliferation inhibition activity (Figure 5) show that all samples A to D can inhibit the proliferation of MDA-MB-175 cells, all of which are dose-dependent, and the proliferation inhibition activity is obviously higher than that of trastuzumab. The activity of KJ015-A and B samples is equivalent to that of the combination of trastuzumab + pertuzumab, the maximum inhibition rate of KJ015-C and D samples is 1.1 times that of the combination of trastuzumab + pertuzumab, and the EC50 value of KJ015-D sample is 1.5μg / ml, which is lower than the 2.3μg / ml of the combination sample of trastuzumab + pertuzumab. The results of BT474 cell proliferation inhibition activity (Figure 6) show that KJ015-A to -D can inhibit the proliferation of BT474 cells, and all of them are dose-dependent. The proliferation inhibition activity of the samples is obviously superior to that of trastuzumab and the combination of trastuzumab and pertuzumab, and the maximum inhibition rate of each is about 1.2 times that of the combination of trastuzumab + pertuzumab. The results of SK-BR-3 cell proliferation inhibition (Figure 7) show that KJ015-A to -D can inhibit the proliferation of SK-BR-3 cells, and all of them are dose-dependent. The proliferation inhibition activity of the samples is obviously superior to that of trastuzumab and the combination of trastuzumab and pertuzumab, and the maximum inhibition rate of each is about 2.2 times that of the combination of trastuzumab + pertuzumab. The results of the NCI-N87 cell proliferation inhibitory activity (Figure 8) show that the killing activity of the four candidate antibodies against NCI-N87 cells is basically equivalent, the maximum killing rate against NCI-N87 cells is all higher than that of trastuzumab and the combination of trastuzumab and pertuzumab, and the maximum inhibition rate is all approximately 1.6 to 17 times that of the combination of trastuzumab + pertuzumab. The results of Calu-3 cell proliferation inhibition (Figure 9) show that KJ015-A to -D can inhibit Calu-3 cell proliferation, and all of them are dose-dependent. The proliferation inhibition activity of the samples is obviously higher than that of trastuzumab, and is about 1.2 times that of the combination of trastuzumab + pertuzumab. Example 15 ADCC activity of bispecific antibodies against different epitopes of human HER2 Place the cell culture flask in a 37°C, 5% carbon dioxide incubator for static culture, observe the color of the medium and cell confluency daily, and record the cell doubling time. Subculture the cells every 2 to 4 days. When the cells fill approximately 80% of the flask, digest and inoculate onto plates. Discard the medium, wash once with PBS, and add 0.25% trypsin (Gibco) for digestion. Pipette the cells into a centrifuge tube and centrifuge at 500 g for 3 min. Discard the supernatant, add complete medium to resuspend the cells, and take 100 μL of the cell suspension for counting. Adjust the cell density to 2.5 to 7.5 × 10 in 2% FBS-containing medium. 5 Adjust the concentration to cells / mL and inoculate into a 96-well plate at 100 μL per well. After overnight culture in a 96-well plate, apply. Dilute the drug to be measured in 1% FBS-containing medium, first dilute the drug to be measured to 75-300 μg / mL (initial concentration can be adjusted according to cells), then dilute 8 times with a 4-5-fold gradient to obtain 9 concentration gradients of the drug, and the last point is the negative control point. The antibody initial concentration varies depending on the cells, and for details, refer to the initial concentration points in the result diagram. Add 25 μL of the diluted drug to the corresponding wells of the 96-well plate in which the cells are inoculated, so that there are two duplicate wells for each point. Incubate the effector cells Jurkat-NFAT / CD16a at 1-3 × 10 in 1640 + 1% FBS. 6 Dilute to 100 µL / mL and add 25 µL to each well (the number of effector cells can be adjusted depending on the cells). After adding sufficient sterile water to the edge of the 96-well plate, place the 96-well plate in a carbon dioxide incubator and incubate for 4 to 6 hours to react. After the incubation is completed, the cells to be detected are removed from the incubator and left at room temperature (22°C to 25°C) for at least 15 min, then the luciferase reagent (Rhinogen®) incubated at room temperature is added to a 96-well plate at 75 μL / well, shaken on a microplate shaker to mix evenly, left at room temperature for 3 to 5 min, and the fluorescence value is read on a microplate reader. Curve fitting analysis is performed on the raw data using GraphPad software. The results of JIMT-1 cell ADCC activity (Figure 10) show that all KJ015-A to D samples have ADCC activity, which is dose-dependent, and the maximum signal value of sample A is 1.2 times the maximum signal value of trastuzumab, which is 1.2 times the maximum signal value when trastuzumab and pertuzumab are used in combination, 1.3 times and 1.2 times when sample B is used, 1.5 times and 1.3 times when sample C is used, and 1.5 times and 1.3 times when sample D is used, indicating that the ADCC activity of KJ015-A to D samples is higher than that of trastuzumab and the combination of trastuzumab and pertuzumab. The results of SK-BR-3 cell ADCC activity (Figure 11) show that all KJ015-A to D samples have ADCC activity that is dose-dependent, and the maximum signal values ​​of samples A to D are 1.8-fold, 1.7-fold, 1.7-fold, and 1.6-fold, respectively, of the maximum signal value obtained when trastuzumab and pertuzumab were used in combination, indicating that the ADCC activity of all KJ015-A to D samples is higher than that obtained when trastuzumab and pertuzumab were used in combination. The results of NCI-N87 cell ADCC activity (FIG. 12) show that all KJ015-A to D samples have ADCC activity and that it is dose-dependent, and the maximum signal values ​​of A to C samples are all about 1.2 times the maximum signal value in the case of the combined use of trastuzumab and pertuzumab, indicating that the ADCC activity of all KJ015-A to C samples is higher than that in the case of the combined use of trastuzumab and pertuzumab. The ADCC activity of the KJ015-D sample is equivalent to that in the case of the combined use of trastuzumab and pertuzumab. The maximum signal value of the activity of the KJ015-E sample is 0.9 times the maximum signal value in the case of the combined use of trastuzumab and pertuzumab, and the EC50 value is 46.07ng / ml, which is clearly different from the 10.17ng / ml in the case of the combined use of trastuzumab and pertuzumab. The results of MDA-MB-175 cell ADCC activity (Figure 13) show that all KJ015-A to D samples have ADCC activity and that it is dose-dependent, and the maximum signal values ​​of A to D samples are about 1.2 times, 1.4 times, 1.3 times, and 1.2 times the maximum signal value in the case of the combination of trastuzumab and pertuzumab, respectively, indicating that the ADCC activity of all KJ015-A to D samples is higher than that in the case of the combination of trastuzumab and pertuzumab. The ADCC activity of the KJ015-E sample is equivalent to that in the case of the combination of trastuzumab and pertuzumab. The sequences primarily involved in the present invention are shown in Table 10 below. (Table 10) Antibody sequences of the present invention JPEG0007644978000011.jpg225144JPEG0007644978000012.jpg87144Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely examples, and that various changes or modifications can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention is determined by the appended claims.

Claims

1. The antibody comprises a first protein domain and a second protein domain for recognizing the extracellular domain II and the extracellular domain IV of HER2, the first protein domain comprises a first heavy chain and a light chain, the second protein domain comprises a second heavy chain and a light chain, the first heavy chain comprises a heavy chain variable region comprising the CDR sequences of HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6-13, and HCDR3 shown in SEQ ID NO: 14-19, the second heavy chain comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 3, or the heavy chain variable region of the second heavy chain comprises a mutation D102E in the amino acid sequence shown in SEQ ID NO: 3, and the light chain comprises a light chain variable region comprising an amino acid substitution at the site of F53 in the amino acid sequence shown in SEQ ID NO: 1; A bispecific antibody against HER2, comprising, in the light chain variable region, one or more amino acid residue substitutions selected from the group consisting of: (1) F53Y; (2) F53Y, L54R, and S56T; (3) N30S and F53Y; and (4) N30S, F53Y, L54R, and S56T in the amino acid sequence shown in SEQ ID NO:

1.

2. The bispecific antibody of claim 1 further comprising a light chain constant region.

3. 3. The bispecific antibody of claim 2, wherein the light chain constant region is a human light chain constant region λ light chain or κ light chain.

4. 3. The bispecific antibody of claim 2, wherein the light chain of the bispecific antibody comprises one or more amino acid residue substitutions in the amino acid sequence set forth in SEQ ID NO:2 selected from the group consisting of: (1) F53Y; (2) F53Y, L54R and S56T; (3) N30S and F53Y; and (4) N30S, F53Y, L54R and S56T.

5. The bispecific antibody of claim 1 , wherein the first heavy chain and the second heavy chain of the bispecific antibody further comprise a heavy chain constant region.

6. The bispecific antibody of claim 5 , wherein the heavy chain constant region is a human heavy chain constant region.

7. The bispecific antibody of claim 5 , wherein the first heavy chain and the second heavy chain of the bispecific antibody form a heterodimer.

8. The bispecific antibody of claim 7 , wherein the first heavy chain and the second heavy chain are connected by a knob-in-hole structure.

9. The heavy chain constant region of the first heavy chain is selected from the group consisting of HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 15, HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 8, HCDR3 shown in SEQ ID NO: 15, HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 9, HCDR3 shown in SEQ ID NO: 15, HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 16, and HCDR1 shown in SEQ ID NO: 5, HCDR3 shown in SEQ ID NO:

16. HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:15, or HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:17, or HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:17, or HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO:17, or HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:13, HCDR3 as shown in SEQ ID NO:17, or HCDR1 as shown in SEQ ID NO:5 2. The bispecific antibody of claim 1 , comprising an HCDR1 as set forth in SEQ ID NO: 5, an HCDR2 as set forth in SEQ ID NO: 7, an HCDR3 as set forth in SEQ ID NO: 18, or an HCDR1 as set forth in SEQ ID NO: 5, or an HCDR2 as set forth in SEQ ID NO: 7, and an HCDR3 as set forth in SEQ ID NO:

19.

10. 2. The bispecific antibody of claim 1 , wherein the heavy chain variable region of the second heavy chain comprises the mutation D102E in the amino acid sequence shown in SEQ ID NO:

3.

11. The bispecific antibody of claim 1, wherein the bispecific antibody has reduced fucose modification.

12. The bispecific antibody of claim 11, wherein the bispecific antibody is a fucose-unmodified antibody.

13. A nucleic acid encoding the bispecific antibody or an antigen-binding portion thereof according to any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid of claim 13.

15. A prokaryotic or eukaryotic cell comprising the expression vector of claim 14.

16. A bispecific antibody-drug conjugate against HER2, comprising the bispecific antibody according to any one of claims 1 to 12 covalently linked to a cytotoxic drug.

17. The bispecific antibody drug conjugate of claim 16, wherein the cytotoxic drug is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA minor groove inhibitor.

18. 18. The bispecific antibody-drug conjugate of claim 17, wherein the tubulin inhibitor is a maytansinoid compound, an auristatin derivative, or tubulin, the topoisomerase inhibitor is DXd or a derivative thereof, SN-38, or adriamycin and a derivative thereof, and the DNA minor groove inhibitor is a calicheamicin, a duocarmycin and a derivative thereof, a pyrrolobenzodiazepine compound, or an indolinobenzodiazepine compound.

19. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 12.

20. 20. The pharmaceutical composition of claim 19, further comprising hyaluronidase.

21. The pharmaceutical composition of claim 20, wherein the hyaluronidase is rHuPH20 or a modified rHuPH20.

22. 20. The pharmaceutical composition of claim 19, further comprising a pharma- ceutically acceptable carrier or excipient.

23. 13. A method for preparing a bispecific antibody according to any one of claims 1 to 12, comprising the steps of: (1) constructing a nucleotide sequence encoding the heavy chain and a nucleotide sequence encoding the light chain in the same vector or different vectors; and (2) expressing the vectors constructed in the steps in different host cells or the same host cell.

24. A medicament for treating a HER2-expressing tumor, the medicament comprising a bispecific antibody against HER2 according to any one of claims 1 to 12, a bispecific antibody-drug conjugate against HER2 according to any one of claims 16 to 18, or a pharmaceutical composition according to any one of claims 19 to 22.

25. The pharmaceutical agent of claim 24, wherein the HER2-expressing tumor comprises breast cancer, gastric cancer, prostate cancer, lung cancer, bladder cancer, ovarian cancer, colon cancer, esophageal cancer, head and neck cancer, endometrial cancer, malignant melanoma, pharyngeal cancer, oral cancer, and skin cancer.

26. The pharmaceutical composition of claim 24, wherein the HER2-expressing tumor is selected from the group consisting of breast cancer, gastric cancer and lung cancer.

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