Anti-HER2 complementary bispecific antibody-drug conjugate as well as preparation method and application thereof

Abstract

The invention provides an anti-HER2 antibody or an antigen binding fragment thereof, an anti-HER2 complementary bispecific antibody and a complementary bispecific antibody-drug conjugate (ADC) constructed by the anti-HER2 antibody or the antigen binding fragment, and a pharmaceutical composition and a kit containing the anti-HER2 antibody or the antigen binding fragment. Also provided are methods of using the antibodies, ADCs, pharmaceutical compositions and kits for treating diseases associated with abnormal expression of HER2.

Description

Anti-HER2 complementary bispecific antibody-drug conjugate and its preparation method and use Technical Field

[0001] The present invention relates to anti-HER2 antibodies or antigen-binding fragments thereof, as well as complementary anti-HER2 bispecific antibodies constructed therefrom and antibody-drug conjugates comprising such antibodies. The present invention also provides methods for preparing the complementary bispecific antibodies and antibody-drug conjugates thereof, as well as methods for treating diseases associated with abnormal HER2 expression. Background Art

[0002] Human Epidermal Growth Factor Receptor 2 (HER2 or ErbB2) is a member of the ErbB / HER family of receptor tyrosine kinases. ErbB / HER family receptors activate their mediated downstream signal transduction pathways through dimerization, thereby participating in the regulation of cell growth, differentiation and survival. HER2 is a transmembrane glycoprotein with a molecular weight of approximately 185 kDa. It consists of an extracellular domain (Extracellular Domain), a transmembrane domain (Transmembrane Domain) and an intracellular tyrosine kinase domain (Intracellular Tyrosine Kinase Domain). ErbB2 / HER2 gene mutations and / or gene amplification can lead to HER2 overexpression, which manifests as IHC 3 in IHC and FISH detection. + or IHC 2 + / FISH + Clinical studies have shown that HER2 is overexpressed in a variety of cancers (such as breast cancer, ovarian cancer, endometrial cancer, gastric cancer, prostate cancer, lung cancer, etc.), and this overexpression is associated with tumor aggressiveness and poor prognosis.

[0003] Several HER2-targeted therapeutic drugs have been approved for the treatment of HER2-overexpressing breast and gastric cancers. These drugs have significantly prolonged patients' survival; however, a considerable number of cancer patients still do not respond to these therapies. In addition, existing HER2-targeted therapies have the following drawbacks that limit their clinical efficacy and lead to safety risks:

[0004] (1) Existing HER2-targeted therapies lack effective killing power against tumor cells with relatively low HER2 expression levels, where the HER2 expression level is 2 in IHC detection. + / FISH - or IHC 1 +(defined as "HER2-low expression"); because HER2 expression levels are highly heterogeneous in HER2-overexpressing tumors, HER2-low-expressing tumor cells in these tumors are easily resistant to existing therapies, resulting in disease progression in most cancer patients after 6 to 12 months of receiving existing HER2-targeted therapies due to drug resistance;

[0005] (2) Compared with HER2 overexpression, approximately 70% of breast cancer patients are characterized by HER2 low expression. Targeting this patient population, DS-8201 is currently the only approved HER2-targeted drug. However, its response rate in the Phase III clinical trial DESTINY-Breast04 was only about 50%, and the incidence of drug-related interstitial lung disease / pneumonitis during the trial was 12.1% (Modi et al., N Engl J Med 2022, 387:9-20). This highlights that there is great room for improvement in the efficacy and safety of existing HER2-targeted therapies in treating HER2-low-expressing tumors.

[0006] (3) The HER2-mediated signal transduction pathway is crucial for maintaining the survival of cardiomyocytes, repairing cardiomyocyte damage, and maintaining the integrity of cardiomyocyte function. Currently approved anti-HER2 antibodies, ADCs, or small molecule inhibitors can block or disrupt the HER2 signal transduction pathway, and therefore have toxic side effects (cardiotoxicity) that can induce cardiac dysfunction. For example, trastuzumab and pertuzumab may both cause a decrease in left ventricular ejection fraction (LVEF) and congestive heart failure during treatment. In particular, trastuzumab has a higher risk of inducing heart failure when administered concurrently with anthracyclines. This leads to the need to strictly screen the population with indications or to provide patients with preventive cardiac protection treatment before using these drugs for treatment.

[0007] Therefore, there is still a large unmet clinical need in this field, and there is an urgent need to develop a targeted drug that is safer (especially to solve the cardiotoxicity problem of existing HER2-targeted therapies) and has stronger killing activity against tumor cells with low HER2 expression, so as to solve the safety and drug resistance problems of existing therapies.

[0008] Summary of the Invention

[0009] The present invention provides a complementary bispecific antibody-drug conjugate targeting HER2 (HER2-targeting Biparatopic ADC). On the one hand, the complementary bispecific ADC can specifically bind to two different epitopes in the extracellular domain of HER2 (also called "extracellular region"), thereby effectively crosslinking the cell surface HER2 to form a matrix-like polymer, thereby inducing rapid and efficient endocytosis, and guiding the endosome after endocytosis from the usual recycling transport pathway to the lysosomal transport pathway. As a result, the ADC bound to the cell surface is efficiently transported (almost 100%) into the lysosome for degradation, thereby releasing more small molecule toxins into the cytoplasm to exert cytotoxicity and subsequent bystander killing effects. Therefore, the complementary bispecific ADC has a broader spectrum of targeted killing activity, which can not only kill HER2-overexpressing cancer cells, but also directly kill HER2-low-expressing (IHC 2 + / FISH - or IHC 1 + ) cancer cells, thereby greatly reducing the chance of drug resistance due to heterogeneity of HER2 expression; on the other hand, the antigen binding epitope of the complementary bispecific ADC is different from that of trastuzumab or pertuzumab, and it will not interfere with the dimerization of HER2 or affect the regulation of HER2 downstream signal transduction pathways, and therefore will not affect the normal biological function of HER2 in cardiomyocytes.

[0010] In one aspect, the present invention provides an isolated anti-HER2 antibody or antigen-binding fragment thereof, wherein the anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the extracellular domain of HER2 and has a high binding activity or binding affinity (e.g., binding affinity constant [K D ] value<1×10 -8 M, <5×10 -9 M, or <1×10 -9 M), and does not cross-bind with other members of the ErbB / HER family (including EGFR, HER3 and HER4). The anti-HER2 antibody or its antigen-binding fragment does not have an inhibitory or activating effect on HER2 and its downstream signal transduction pathways.

[0011] In some embodiments, the anti-HER2 antibody comprises a murine anti-HER2 antibody or a chimeric antibody and / or a humanized antibody derived therefrom and an optimized antibody thereof. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment specifically binds to subdomains 1, 3, and / or 4 of the HER2 extracellular region, preferably subdomain 1 or 3 of the HER2 extracellular region. In some embodiments, the two anti-HER2 antibodies or their antigen-binding fragments used to construct the anti-HER2 complementary bispecific antibody specifically recognize different epitopes in the HER2 extracellular region, and there is no competitive binding relationship between them.

[0012] On the one hand, the present invention provides an anti-HER2 complementary bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first and second antigen-binding domains specifically bind to different epitopes of HER2, resulting in the bispecific antibody being able to cross-link HER2 on the cell surface to form clusters, and the formation of clusters is subject to the abundance of HER2 expression on the cell surface; the formation of clusters triggers the rapid endocytosis of HER2 by the cell and its transport to the lysosome for degradation. Compared to the monospecific anti-HER2 antibodies and / or trastuzumab described in the present invention, the complementary bispecific antibody has a significantly improved HER2 internalization rate and a higher lysosomal transport / degradation efficiency.

[0013] In some embodiments, the first and second antigen binding domains of the anti-HER2 complementary bispecific antibody specifically bind to different (non-overlapping or non-competing) epitopes of HER2, and the epitopes include sequences located in HER2 extracellular region subdomains 1, 3 and / or 4. In one embodiment, the first antigen binding domain specifically binds to HER2 extracellular region subdomain 3, and the second antigen binding domain specifically binds to HER2 extracellular region subdomain 1, and the epitopes bound are different from the antigen binding epitopes of Trastuzumab or Pertuzumab. In one embodiment, the complementary bispecific antibody has no inhibitory or activating effect on cell surface HER2 and its downstream signal transduction pathways. In one embodiment, the complementary bispecific antibody triggers its rapid internalization by the cell and efficient lysosomal transport after binding to and cross-linking HER2 on the cell surface. In one embodiment, the complementary bispecific antibody can induce degradation of cell surface HER2. In one embodiment, the complementary bispecific antibody cross-links cell surface HER2, causing it to be internalized and transported into lysosomes for degradation, resulting in a significant decrease in the abundance of cell surface HER2, thereby effectively inhibiting the proliferation of HER2-overexpressing tumor cells.

[0014] On the one hand, the present invention provides an ADC, which couples a small molecule toxin compound to the anti-HER2 complementary bispecific antibody of the present invention via a linker. The ADC can specifically bind / cross-link HER2 expressed on the surface of tumor cells and aggregate into "clusters", so that the ADC is rapidly internalized and transported to lysosomes for degradation, significantly improving the efficiency of the small molecule toxin compound in the ADC entering tumor cells and being released within tumor cells. Therefore, the complementary bispecific ADC has a broader spectrum of tumor cell killing activity than T-DM1 and / or DS-8201, that is, the ADC has direct killing activity against both HER2-overexpressing and HER2-low-expressing tumor cells. The ADC can be represented by formula (I): Ab-(LD)p (I)

[0015] wherein Ab represents the anti-HER2 complementary bispecific antibody of the present invention;

[0016] D represents a small molecule toxin compound (Drug);

[0017] L represents a cleavable linker that couples Ab to D; and

[0018] p represents the copy number of (LD) coupled to the Ab, ranging from 2 to 8.

[0019] In some embodiments, the small molecule toxin compound includes cytotoxins and chemotherapeutic drugs. In one embodiment, the small molecule toxin compound is a cytotoxin, including a microtubule inhibitor and a DNA damaging agent; preferably, the microtubule inhibitor includes Eribulin, Auristatins derivatives (e.g., MMAE, MMAF, MMAD), Tubulysins, Cryptomycins and Maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4), and the DNA damaging agent includes a topoisomerase inhibitor (e.g., camptothecin derivative SN-38, Exatecan and DXd [Exatecan derivative for ADC]), pyrrolobenzodiazepine (PBD), calicheamicin and its derivatives (e.g., N-acetylcalicheamicin [CMC]), and duocarmycin. In one embodiment, the small molecule toxin compound is Eribulin.

[0020] In some embodiments, the cleavable linker can be any linker comprising a cleavable portion, and the cleavable portion comprises any cleavable chemical bond. In some specific embodiments, the cleavable linker comprises a cleavable peptide portion that can be cleaved by an intracellular peptidase or protease, and the cleavable peptide portion comprises an amino acid unit, and the amino acid unit comprises a dipeptide, a tripeptide, or a tetrapeptide. In some specific embodiments, the cleavable linker may comprise at least one spacer (Spacer) for conjugating the anti-HER2 complementary bispecific antibody (Ab) of the present invention to a small molecule toxin compound (D), the spacer comprising a spacer conjugated to the antibody, and / or a second spacer conjugated to the small molecule toxin compound. In some embodiments, the spacer conjugated to the antibody is hydrophilic, and an exemplary spacer comprises polyethylene glycol (PEG). In some embodiments, the spacer is connected to the anti-HER2 complementary bispecific antibody of the present invention via butylene diimide (Mal). In some embodiments, the second spacer conjugated to the small molecule toxin compound is used to connect the cleavable portion of the cleavable linker (e.g., a cleavable peptide) to the small molecule toxin compound. In some embodiments, the second spacer conjugated to the small molecule toxin compound has the property of self-immolation, and the self-immolation spacer comprises a p-aminobenzyl unit. In some embodiments, the cleavable linker comprises a Mal-spacer and a cleavable peptide portion. In some embodiments, the Mal-spacer of the cleavable linker is coupled to one or more amino acid residues of the antibody portion in the ADC of the present invention. In some embodiments, the maleimide group of the Mal-spacer can react with the sulfhydryl group of the cysteine ​​residue at a specific position in the constant region and / or variable region of the antibody. In some embodiments, the second spacer of the cleavable linker is coupled to the small molecule toxin compound portion of the ADC of the present invention, and the small molecule toxin compound is eribulin or a derivative thereof. In some embodiments, the cleavable portion of the cleavable linker (e.g., a cleavable peptide) can be directly conjugated to the small molecule toxin compound portion of the ADC, and the small molecule toxin compound is eribulin or a derivative thereof.

[0021] In some embodiments, p is 2 to 8, for example, 4 to 8. Herein, p may be an integer greater than 0 or a non-integer.

[0022] In another aspect, the present invention relates to isolated nucleic acid molecules (also referred to as "polynucleotides") encoding anti-HER2 complementary bispecific antibodies and their corresponding monospecific antibodies or antigen-binding fragments thereof, as well as expression vectors comprising the nucleic acids and host cells comprising the nucleic acids or expression vectors. The present invention also relates to methods for using the host cells to prepare the monospecific anti-HER2 antibodies or antigen-binding fragments thereof and the anti-HER2 complementary bispecific antibodies described herein, the methods comprising culturing the host cells and recovering the antibodies or antigen-binding fragments thereof from the culture medium.

[0023] In another aspect, the present invention relates to a pharmaceutical composition comprising the monospecific anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody, or the anti-HER2 complementary bispecific antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.

[0024] In another aspect, the present invention relates to a drug kit comprising an effective amount of the anti-HER2 monospecific antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, the anti-HER2 complementary bispecific antibody-drug conjugate, or the pharmaceutical composition of the present invention, and optionally at least one additional tumor therapeutic agent.

[0025] In another aspect, the present invention relates to a method for treating a HER2-expressing tumor in a subject, the method comprising administering to a subject in need thereof an anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition, or a kit of the present invention. Alternatively, the present invention relates to the use of the anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition, or a kit of the present invention for the preparation of a medicament for treating a HER2-expressing tumor in a subject. Alternatively, the present invention relates to the anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition, or a kit of the present invention for treating a HER2-expressing tumor in a subject. In some embodiments, the tumor comprises HER2 overexpression (IHC 3 + , or IHC 2 + / FISH + ) tumors and / or low HER2 expression (IHC 2 + / FISH - , or IHC 1 +) tumors. In some embodiments, the anti-HER2 complementary bispecific antibody-drug conjugate or its pharmaceutical composition has a killing effect on tumor cells that overexpress and / or underexpress HER2. In some embodiments, the subject is a mammal. In some embodiments, the tumors include breast cancer, ovarian cancer, cervical cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, bladder cancer, melanoma, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, endometrial cancer, etc., and the tumors can overexpress or underexpress HER2.

[0026] In some embodiments, the tumor is a tumor that is resistant to existing HER2 targeted therapeutics. In some embodiments, the tumor is a tumor that is resistant to HER2 targeted therapeutics including Trastuzumab, Pertuzumab, T-DM1 and / or DS-8201. In one embodiment, the tumor is a tumor that does not respond or responds poorly to targeted therapeutics including Trastuzumab, Pertuzumab, T-DM1, DS-8201 and / or taxanes (e.g., Paclitaxel, Docetaxel, Cabazitaxel, etc.).

[0027] In some embodiments, the subject includes patients who are not suitable for or are refractory to existing HER2 targeted therapies, or who develop resistance or relapse after receiving existing HER2 targeted therapies. In some embodiments, the existing HER2 targeted therapies include treatment with trastuzumab, pertuzumab, T-DM1, and / or DS-8201.

[0028] In another aspect, the present invention relates to a method for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, or a method for screening cancer patients who are responsive to treatment with the anti-HER2 complementary bispecific ADC described in the present invention, comprising incubating the anti-HER2 monospecific antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody described in the present invention with the sample or a biological sample isolated from the patient, and detecting whether the antibody binds to the sample or biological sample.

[0029] The present invention will be further illustrated by the following drawings and specific embodiments, and other features and advantages disclosed by the present invention will be apparent. These drawings and specific embodiments should not be considered to limit the scope of the present invention, and modifications that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the scope of protection of the appended claims. All references cited in this invention, including publications, patents, and patent applications, are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1. ELISA was used to detect the binding activity of various anti-HER2 chimeric antibodies to human ErbB / HER family proteins EGFR, HER2, HER3, and HER4. The control antibodies included trastuzumab, cetuximab, and patritumab.

[0031] Figure 2. Flow cytometry analysis of the binding activity of anti-HER2 chimeric antibodies to HER2-overexpressing NCI-N87 cells, where trastuzumab was used as a control antibody.

[0032] Figure 3. Schematic diagram of the structure of the human-mouse chimeric HER2 extracellular region recombinant protein (Figure 3A), and the HER2 extracellular region subdomain where the antigen-binding epitope of the anti-HER2 chimeric antibody is located determined by ELISA (Figure 3B).

[0033] Figure 4. Competition ELISA was used to detect whether there was a competitive binding relationship between the anti-HER2 chimeric antibodies or with the control antibody trastuzumab. Figure 4A shows the results of biotin-labeled mAb2164 competing with mAb2117, mAb2126, mAb2170, and trastuzumab for binding to HER2 recombinant protein, respectively. Figure 4B shows the results of mAb2117 competing with biotin-labeled mAb2128 or biotin-labeled mAb2126 for binding to HER2 recombinant protein, and mAb2164 competing with biotin-labeled mAb2128 for binding to HER2 recombinant protein.

[0034] Figure 5. Effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on phosphorylation of HER2 intracellular domain Y1248 in SKBR-3 cells, wherein control antibodies include trastuzumab and pertuzumab.

[0035] Figure 6. Effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on NRG-1-induced AKT phosphorylation in T47D cells, wherein control antibodies include trastuzumab and pertuzumab.

[0036] Figure 7. Flow cytometry was used to detect the binding activity of optimized anti-HER2 single-chain antibody molecules containing single / multi-point mutations to BT474 cells (Figure 7A) and RT-112 cells (Figure 7B), respectively, where the control antibody was the parent single-chain antibody Hu2117HK.

[0037] Figure 8. Flow cytometry was used to detect the binding activity of optimized anti-HER2 single-chain antibody molecules containing combined mutations (Hu2117-HK203, Hu2117-HK303, Hu2117-HK304, Hu2117-HK309, Hu2117-HK310) to BT474 cells and RT-112 cells, respectively, where the control antibody was the parent single-chain antibody Hu2117HK.

[0038] Figure 9. Schematic diagram of the structure of an exemplary anti-HER2 complementary bispecific antibody 04BS-109-WT. The configuration of the exemplary complementary bispecific antibody is a DVD-IgG structure, wherein the Fv domain is the variable region sequence of Hu2117-HK304, the IgG domain is the full-length sequence of Hu2126-H2K1-L71-H72b-Mu14, the Fv domain heavy chain variable region is connected to the heavy chain of the IgG domain via a linker (G4S) 1, and the Fv domain light chain variable region is connected to the light chain of the IgG domain via a linker (G4S) 3. The antibody heavy chain Fc region contains L234F / L235E / P331S (EU Numbering) mutations.

[0039] Figure 10. Flow cytometry was used to determine the co-binding or competitive binding relationship between the anti-HER2 humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) and BT474 cells.

[0040] Figure 11. Flow cytometry was used to detect the endocytosis reaction of the anti-HER2 complementary bispecific antibody 04BS-109-WT and its mutant molecules in cell lines expressing different levels of HER2 (BT474, JIMT-1, RT-112), wherein the control antibodies included Trastuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibody 04BS-1123-ST06.

[0041] Figure 12. Confocal microscopy was used to detect the lysosomal transport efficiency of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 after endocytosis by SKBR-3 cells, where control antibodies included trastuzumab and human IgG isotype antibodies; the arrows in the figure indicate that the fluorescent signal sites of the antibody and the fluorescent signal sites of the lysosome overlap with each other, indicating that the cross-linked aggregates or clusters formed by the complementary bispecific antibody and HER2 are endocytosed and transported into lysosomes; T represents the antibody incubation time (hours).

[0042] Figure 13. Western-blot analysis of HER2 protein degradation in BT474 cells induced by the anti-HER2 complementary bispecific antibody 04BS-1123-ST06. Control antibodies included trastuzumab and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (Hu2126-7172b-Mu14 for short), corresponding to the complementary bispecific antibody.

[0043] Figure 14. Effect of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on the in vitro proliferation of BT474 cells, wherein the control antibodies include Trastuzumab, Pertuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibodies.

[0044] Figure 15. Reporter gene assay was used to detect the effects of anti-HER2 monospecific antibodies (Figure 15A) and anti-HER2 complementary bispecific antibodies (Figure 15B) on NRG-1-induced HER2 / HER4 dimerization, where control antibodies included trastuzumab and pertuzumab.

[0045] Figure 16. Western-blot analysis of the effect of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes. Control antibodies included trastuzumab and pertuzumab.

[0046] Figure 17. ADCC activity of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 was detected using a reporter gene assay, wherein the positive control antibody was trastuzumab.

[0047] Figure 18. ELISA was used to detect the binding specificity of the anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) to human ErbB / HER family members (including EGFR, HER2, HER3 and HER4).

[0048] Figure 19. In vitro cell killing assay was used to detect the proliferation inhibitory activity of anti-HER2 complementary bispecific ADCs (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) and the benchmark molecule DS-8201 in tumor cell lines expressing different levels of HER2.

[0049] Figure 20. In vitro cell cytotoxicity assay for the bystander effect of an anti-HER2 complementary bispecific ADC. Figure 20A shows the results of an in vitro cell cytotoxicity assay for BT474 cells treated with ST06-GGFG-Eribulin for 3 days. Simultaneously, the culture supernatant (BT474-conditioned medium) was collected and used to incubate MDA-MB-468 cells. Three days later, the survival of the MDA-MB-468 cells was assessed to determine the bystander cytotoxicity of the ADC. A control group showed the survival of MDA-MB-468 cells incubated simultaneously with freshly prepared ST06-GGFG-Eribulin. Figure 20B shows the cytotoxicity of ST06-GGFG-Eribulin against HER2-overexpressing BT474 cells and HER2-non-expressing Jurkat cells, measured by flow cytometry, when cultured alone or in co-culture. The numbers shown in the upper left and lower right corners of the figure are the viable cell counts for BT474 and Jurkat cells, respectively.

[0050] Figure 21. The in vivo anti-tumor activity of the anti-HER2 complementary bispecific ADC (ST06-GGFG-Eribulin) was detected in mouse subcutaneous xenograft tumor models constructed based on the tumor cell lines NCI-N87 (Figure 21A), JIMT-1 (Figure 21B) and RT-112 (Figure 21C). The control groups included a mixture of antibodies and small molecule compounds (ADMix), a DS-8201 group, and a vehicle group (Vehicle). All tumor-bearing mice were injected with the drug via tail vein. The arrows in the figure indicate the time points of administration.

[0051] Figure 22. The in vivo anti-tumor activity of the anti-HER2 complementary bispecific ADC (ST06-GGFG-Eribulin) was tested using a mouse subcutaneous xenograft tumor model with acquired resistance to DS-8201, where the control groups included the DS-8201 group and the vehicle group (Vehicle).

[0052] Detailed Description of the Invention

[0053] definition

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0055] As used herein, the terms "HER2" and "HER2 receptor" are used interchangeably, and the protein is also referred to as ErbB2, c-ERB2, c-ERB-2, NEU, HER-2 / neu, p185 (erbB2), or CD340. Unless otherwise indicated, it is from a non-human species, e.g., "mouse HER2," "monkey HER2," etc., as used herein, refers to any native form of human HER2, which may have an amino acid sequence as shown in SEQ ID NO: 234 and / or a full-length HER2 amino acid sequence as shown in NCBI Accession No. NP_004439.2, or may be naturally expressed by cells (including tumor cells) or expressed by cells transfected with a HER2 gene or cDNA. The term includes naturally occurring HER2 allelic variants and splice variants, isoforms, homologs, and species homologs. HER2 can be isolated from the human body or produced by recombinant or synthetic methods.

[0056] The extracellular domain of HER2 is composed of four subdomains, namely subdomain 1 (D1, approximately amino acid residues 1-195), subdomain 2 (D2, approximately amino acid residues 196-319), subdomain 3 (D3, approximately amino acid residues 320-488), and subdomain 4 (D4, approximately amino acid residues 489-630) (residue numbering without signal peptide); among them, D2 and D4 are cysteine-rich domains responsible for receptor dimerization (Garrett et al., Mol Cell 2003, 11:495-505; Cho et al., Nature 2003, 421:756-760; Franklin et al., Cancer Cell 2004, 5:317-328).

[0057] Herein, "cells expressing HER2" may be naturally occurring cells or cell lines (eg, tumor cells), or may be recombinantly produced by introducing a nucleic acid encoding HER2 into a host cell.

[0058] As used herein, the term "bispecific antibody" or "bispecific antibody" is intended to include any antibody or antigen-binding fragment that specifically binds to two different antigenic epitopes, comprising two independent antigen-binding domains, each with a unique antigen-binding specificity. For example, the term "complementary bispecific antibody" as used herein specifically refers to a class of bispecific antibodies in which the first and second antigen-binding domains bind to different epitopes on the same antigen.

[0059] A monospecific antibody is an antibody or antigen-binding fragment that has only one binding specificity, i.e., the antigen-binding domain of a monospecific antibody binds to a single epitope of a single antigen. In some embodiments, examples of monospecific antibodies include the anti-HER2 monospecific antibodies of the present invention.

[0060] As used herein, "antigen-binding domain," "antigen-binding region," "epitope-binding domain," or "antigen-binding polypeptide" are used interchangeably and refer to a specific region of an antibody, antigen-binding fragment, or derivative thereof that is directly involved in a specific interaction with a target antigen, such as by binding, steric hindrance, stabilization / destabilization, spatial distribution, or the like, to achieve dynamic equilibrium with the target antigen. In the present invention, "antigen-binding domain" also refers to a specific region of the antibody, antigen-binding fragment, or derivative thereof that interacts with a specific epitope on HER2, achieving dynamic equilibrium between the two by binding, steric hindrance, stabilization / destabilization, spatial distribution, or the like.

[0061] "Antibody" refers to a polypeptide or protein that is generally encoded by an immunoglobulin gene or multiple immunoglobulin genes, or fragments thereof, and is capable of specifically recognizing and binding an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin class or isotype: IgG, IgM, IgA, IgD, and IgE, respectively, several of which can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The typical immunoglobulin (e.g., antibody) structural unit is a tetramer, each composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of each chain defines a variable (V) region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region typically includes three domains, CH1, CH2, and CH3. The light chain consists of a light chain variable region (VL) and a light chain constant domain (CL), wherein the light chain constant domain typically includes one domain, CL. The pairing of VH and VL together forms a single antigen-binding site. Endogenous VL is encoded by gene segments V (variable) and J (joining), and endogenous VH is encoded by V, D (diversity), and J. Both VL and VH include hypervariable regions (also known as complementarity determining regions (CDRs) and framework regions (FRs). The terms "variable region" or "V region" are used interchangeably and refer to the heavy chain variable region or light chain variable region arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from amino terminus to carboxyl terminus. The term "J region" refers to the subsequence of the variable region encoding the C-terminal portion containing CDR3 and FR4. The V region or J region can be naturally occurring, recombinant or synthetic. Herein, the antibody light chain variable region and / or the antibody heavy chain variable region are sometimes collectively referred to as "antibody variable region", and the antibody light chain and / or the antibody heavy chain are collectively referred to as "antibody chain". In certain embodiments, the FRs of the antibodies or antigen-binding fragments thereof provided herein may be identical to human germline sequences, or may be natural or artificially modified.

[0062] The positions of CDRs and FRs can be determined using various definitions well known in the art, for example, Kabat, Chothia, IMGT, and Contact (see: Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 1991, NIH Publication No. 91-3242; Johnson et al., Nucleic Acids Res 2001, 29:205-206; Chothia & Lesk, J Mol Biol 1987, 196:901-917; Chothia et al., Nature 1989, 342:877-883; Chothia et al., J Mol Biol 1992, 227:799-817; Al-Lazikani et al., J Mol Biol 1997, 273:927-748; Lefranc et al., Nucleic Acids Research 2001, 34:106-111; 1999, 27:209–212; MacCallum et al., J Mol Biol 1996, 262:732-745). The definition of antigen binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefranc, Nucleic Acids Res 2001, 29:207-209; Lefranc, The Immunologist 1999, 7:132-136; Lefranc et al., Dev Comp Immunol 2003, 27:55-77; MacCallum et al., J Mol Biol 1996, 262:732-745; Martin et al., Proc Natl Acad Sci USA 1989, 86:9268-9272; Martin et al., Methods Enzymol 1991, 203:121-153; Sternberg, ed., Protein Structure Prediction: A Practical Approach, 1996 Oxford University Press. The present invention encompasses any definition method for determining the CDRs in the anti-HER2 complementary bispecific antibodies or anti-HER2 antibodies or antigen-binding fragments thereof of the present invention. Table 1 shows the position numbering of the antibody CDR amino acid sequences determined using different definition methods. The exact number of amino acid residues encompassing a particular CDR varies depending on the sequence of the CDR.When the amino acid sequence of the antibody variable region is known, those skilled in the art can determine the CDR of the antibody by conventional methods including but not limited to the above definitions.

[0063] Table 1. CDRs determined by different definition methods 1 1 The numbering of all CDR definitions in Table 1 follows the numbering system proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, NIH Publication No. 91-3242).

[0064] In addition, Kabat et al. also defined a numbering system for variable region sequences that can be applied to any antibody. A person of ordinary skill in the art can clearly apply this "Kabat numbering" system to the variable region sequence of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence. Unless otherwise indicated, the numbering of specific amino acid residue positions in the variable region of the antigen-binding domain of the anti-HER2 antibody, anti-HER2 complementary bispecific antibody, or complementary bispecific ADC described in the present invention is determined according to the Kabat numbering system.

[0065] Antibodies exist in the form of complete immunoglobulins, or exist in the form of many fragments produced by digestion with a variety of peptidases. Although various antibody fragments have been defined according to the digestion of complete antibodies, it will be appreciated by the skilled artisan that such fragments can be synthesized by chemical cleavage methods or by using recombinant DNA methods. In this article, the "antigen binding fragment" (or simply "antibody portion" or "antibody fragment") of the term antibody refers to an antibody portion containing one or more CDRs or other any antibody fragments that can be combined with an antigen (for example, the extracellular domain of HER2 or HER2) but do not have the structure of a complete antibody. Antigen binding fragments can have the same activity of specifically binding antigens as complete antibodies. Preferred antigen binding fragments also retain the ability to internalize into cells expressing the target antigen. In certain embodiments, an antigen binding fragment can contain one or more CDRs from a specific human antibody, grafted to the framework region from one or more different human antibodies. Antigen-binding fragments include, but are not limited to: (i) a "Fab" fragment, a monovalent antibody fragment consisting of the VH, VL, CL, and CH1 domains; (ii) a "F(ab')2" fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an "Fv" fragment, consisting of the VL and VH domains of a single antibody arm and the smallest antibody fragment containing a complete antigen-binding site; (iv) an "Fd" fragment, consisting of the VH and CH1 domains; (v) a "single-chain Fv antibody (scFv)" or "single-chain antibody," which refers to an engineered antibody in which the variable region of the light chain is linked directly to the variable region of the heavy chain or connected by a peptide chain (Huston et al., Proc Natl Acad Sci USA 1988, 85:5879-5883; Bird et al., Science 1988, 242: 423-426), such scFv antibodies can be internalized into cells when bound to cell surface antigens (He et al., J Nucl Med 2010, 51: 427-432; Fitting et al., MAbs 2015, 7: 390-402); In addition, single-chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions (Zapata et al., Protein Eng 1995, 8: 1057-1062; US5641870); (vi) "dAb" fragments (Ward et al., Nature 1989, 341: 544-546; WO90 / 05144A1) comprise a single variable domain, such as a VH domain.Single-domain antibodies (sdAbs) are independent immunoglobulin domains; (vii) “Diabodies” are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thus forcing the two domains to pair with complementary domains from another chain to form two antigen-binding sites (Holliger et al., Proc Natl Acad Sci USA 1993, 90:6444-6448; Poljak et al., Structure 1994, 2:1121-1123; EP 404097; WO 93 / 11161).

[0066] As used herein, the term "Fc region" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region, such as an immunoglobulin heavy chain constant region in addition to the first constant region (CH1). For IgG, the Fc region may include immunoglobulin domains CH2 and CH3 and a hinge region between CH1 and CH2. The Fc region used herein includes native sequence Fc regions and / or Fc region variants, and may be a part of an anti-HER2 antibody, an anti-HER2 complementary bispecific antibody, or an ADC thereof of the present invention. It should be understood in the art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is generally defined as comprising a cysteine ​​residue at position 226 or a proline residue at position 230 at its amino terminus, according to the EU numbering system / scheme, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242.

[0067] The term "anti-HER2 antibody" or "antibody that specifically binds to HER2" refers to any form of antibody or fragment thereof that specifically binds to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as the fragment specifically binds to HER2.

[0068] As used herein, the term "specific binding" or "binding specificity" or "specific for" or "binding" refers to a binding reaction that determines the presence of a target molecule (e.g., an antigen) in a heterogeneous population of proteins and other biological products (e.g., biological samples such as blood, serum, plasma, or tissue samples), that is, the binding is selective for the target molecule and can distinguish between undesirable or non-specific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) binds to the target molecule with higher affinity, stronger binding activity, greater ease of binding, and / or longer duration of binding than the antibody binds to other non-target molecules. Thus, as desired, this selection can be achieved by removing antibodies that cross-bind to other ErbB / HER family members. Various immunoassays can be used to select antibodies that specifically react with a particular protein, such as ELISA assays. Typically, under defined assay conditions, the specific or selective binding reaction of an antibody or binding agent to an antigen will produce a signal at least twice the background, more typically at least 10-100 times the background, and will not substantially bind to other antigens present in the sample in significant amounts. In certain embodiments, the equilibrium dissociation constant (K) for specific binding of an antibody to a target antigen is D ) is <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0069] In this article, the term "monoclonal antibody" refers to an antibody obtained from an antibody population that is substantially homogeneous, that is, each antibody in the antibody population is identical except for the natural mutations that may be present in small amounts. Monoclonal antibodies exhibit a high degree of binding specificity and affinity for a specific epitope. Monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al., Nature 1975, 256: 495, or by recombinant DNA methods (see US4816567). They can also be isolated from phage antibody libraries, for example, with reference to Clackson et al., Nature 1991, 352: 624-628; Marks et al., J Mol Biol 1991, 222: 581-597.

[0070] As used herein, the term "chimeric antibody" refers to an antibody (e.g., US4816567) that contains sequences derived from two different antibodies, typically derived from different species. For example, a chimeric antibody comprises human and rodent antibody fragments, typically a human constant region and a mouse variable region. Methods for producing chimeric antibodies include recombinant DNA and gene transfection techniques known to those of ordinary skill in the art (e.g., Morrison et al., Proc Natl Acad Sci USA 1984, 81: 6851-6855; US 5202238 and US 5204244).

[0071] As used herein, the term "humanized antibody or antigen-binding fragment" refers to an antibody or antigen-binding fragment that includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans. A humanized antibody optionally also includes at least a portion of a constant region of a human immunoglobulin. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for administration to humans. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, except for the CDR sequences that are non-human, the rest of the antibody is essentially composed of human sequences. In some embodiments, humanized antibodies can be further modified, improved, and optimized for specificity, affinity, and / or activity by replacing corresponding residues in the human immunoglobulin FR framework region with residues from antibodies of non-human species. In some embodiments, the FR region derived from humans may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, for example, no more than 5, 4, 3, 2, or 1 amino acid changes. In some embodiments, the amino acid changes can occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0072] As used herein, the term "corresponding human germline sequence" refers to an antibody variable region amino acid sequence or subsequence that, when compared to all other known human germline immunoglobulin variable region amino acid sequences, has the highest amino acid sequence identity to a reference human germline immunoglobulin variable region amino acid sequence. A corresponding human germline sequence can be a framework region alone, a complementarity determining region alone, a framework region and complementarity determining regions, a variable region, or other combinations comprising variable region sequences or subsequences. Sequence identity can be determined using methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or other alignment algorithms known in the art. A corresponding human germline amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference human germline immunoglobulin variable region amino acid sequence.

[0073] The antibody portion of the anti-HER2 antibody, anti-HER2 complementary bispecific antibody or ADC of the present invention can be selected from any one or more of the following forms, including chimeric form, non-human form, humanized form or fully human form, as long as the form can specifically bind to HER2.

[0074] As used herein, the term "epitope" refers to a protein determinant, the portion of an antigen that can be recognized and specifically bound by an antibody. An epitope is typically composed of surface groups of a molecule, such as amino acids or sugar side chains, and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. The portion of an antibody or its antigen-binding fragment that recognizes the epitope is called a paratope.

[0075] Competitive binding and epitope grouping can also be used to determine whether different antibodies bind to the same epitope or overlapping epitopes, for example, using the method described in Harlow and Lane (eds., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory). In some embodiments, the activity of an antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment comprising a CDR and / or variable region selected from those identified in Tables 2 and 3) and other antibodies or antigen-binding fragments thereof in inhibiting each other's binding to a target antigen (e.g., HER2) is detected by competitive binding ELISA, and when the binding activity is reduced by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, or any percentage between the recited values), the two are determined to be competitively bound, i.e., bound to the same epitope or overlapping epitopes. In some embodiments, competitive binding can be attributed to shared (Shared) or similar (e.g., partially overlapping) epitopes, or to steric hindrance caused by the binding of antibodies or antigen-binding fragments to nearby epitopes (see Morris, ed., Methods in Molecular Biology 1998, Vol. 66, pp. 55-66). In some embodiments, competitive binding can be used to group antibodies or antigen-binding fragments that bind to a shared similar epitope, for example, antibodies or antigen-binding fragments that compete for binding can be "grouped" into a group of antibodies or antigen-binding fragments with overlapping or proximal epitopes, while non-competing antibodies or antigen-binding fragments are grouped into another group of antibodies or antigen-binding fragments that do not have overlapping or proximal epitopes.

[0076] The term "affinity" or "binding affinity" refers to the intrinsic binding ability of an interaction between a molecule (e.g., a receptor or antigen) and its partner (e.g., a ligand or antibody), i.e., the strength of the sum of all non-covalent interactions. Unless otherwise stated, "binding affinity" as used herein is a term reflecting the intrinsic binding ability of a one-to-one interaction between members of a binding pair (e.g., a receptor and a ligand or an antigen and an antibody). The affinity of a molecule X for its partner Y can generally be expressed in terms of the equilibrium dissociation constant (K D ) indicates that the equilibrium dissociation constant is the dissociation rate constant (k dis or k off ) and the binding rate constant (k a or k on Affinity can be measured by common methods known in the art, including the methods used in the present invention.

[0077] As used herein, "internalization" or "endocytosis" refers to the ability of an antibody, antigen-binding fragment, bispecific antibody, or ADC, when bound to a cell surface target antigen, to enter the cell's endosomes through the cell's lipid bilayer membrane (i.e., endocytosis), preferably into the cell's lysosomes. In some embodiments, the anti-HER2 complementary bispecific antibody or ADC described herein, after binding to HER2 on the tumor cell membrane, can be internalized into the cell and downregulate HER2 expression on the tumor cell surface.

[0078] In this article, the term "antibody variant" or "antibody variant" refers to an antibody polypeptide sequence containing at least one amino acid mutation in the original antibody variable region. Variant can be substantially homologous or substantially identical to unmodified antibodies. In some embodiments, one, two, three, four, five and / or six CDRs of the antibody portion of an anti-HER2 antibody of the present invention, an anti-HER2 complementary bispecific antibody or an ADC are provided to improve and optimize the performance of the antibody or Fab, including but not limited to improving the degree of humanization, enhancing binding affinity or binding activity to HER2, improving internalization efficiency, increasing expression, and / or improving stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, amino acid mutations are present in one, two, three, and / or four FRs of the anti-HER2 antibodies, anti-HER2 complementary bispecific antibodies, or ADC antibody portions of the present invention to improve and optimize the performance of the antibody or antigen-binding portion, including but not limited to increasing the degree of humanization of the antibody or antigen-binding portion, enhancing binding affinity or binding activity to HER2, increasing expression levels, and / or increasing stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, one or more amino acid mutations are made in the CDRs and FRs of the anti-HER2 antibodies, anti-HER2 complementary bispecific antibodies, or ADC antibody portions of the present invention to increase the degree of humanization of the antibody or antigen-binding fragment, enhance binding affinity or binding activity to HER2, increase internalization rate, increase expression levels, and / or increase stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, the amino acid mutations comprise amino acid substitutions, removals, insertions, or any combination thereof.

[0079] Among them, amino acid substitutions include conservative amino acid substitutions and non-conservative amino acid substitutions. Conservative amino acid substitutions involve replacing an amino acid with another amino acid of the same class (similar in chemical properties or functions), while non-conservative substitutions involve replacing an amino acid with an amino acid of a different class (dissimilar in chemical properties or functions). One of ordinary skill in the art can perform conservative or non-conservative amino acid substitutions based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, (i) non-polar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W) and methionine (Met, M); (ii) polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln, Q); (iii) positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K) and histidine (His, H); (iv) negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Generally speaking, conservative amino acid substitutions do not substantially alter the functional properties of a protein, whereas non-conservative amino acid substitutions may significantly alter the properties or functions of a protein. Although the site or region for introducing an amino acid sequence mutation can be predetermined, the potential changes in protein properties or functions brought about by non-conservative substitutions are unpredictable.

[0080] As used herein, the terms "identical" or "identity" or "percentage identity" or "percentage sequence identity" are used interchangeably for two or more polypeptide sequences and refer to the percentage ratio of the number of amino acid residues in a candidate sequence that are identical to a reference sequence in the total number of amino acid residues in the candidate sequence, after introducing the necessary intervals to maximize the number of identical amino acids. Conservative substitutions of the amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid sequences using tools disclosed in the art, such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266: 383-402; Larkin et al., Bioinformatics 2007, 23: 2947-2948) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tools or appropriately adjust the parameters as needed for comparison, for example, by selecting a suitable algorithm for sequence alignment.

[0081] In this article, when it comes to protein, the term "isolated" means that the protein is substantially free of other cellular components that are bound to it in the native state, preferably in a homogeneous state, for example, an isolated protein can be removed from a natural or natural environment. The isolated protein can be lyophilized or an aqueous solution. Generally, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). In the isolated preparation, the protein is substantially purified. The term "purified" means that the protein essentially produces only one band in a non-reducing electrophoresis gel. In particular, this means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. With regard to the present invention, in some embodiments, recombinant proteins expressed in host cells are considered to be isolated, and the same is also applicable to natural or recombinant proteins that are separated, fractionated, or partially or substantially purified by any technology well known to those skilled in the art. In some embodiments, " isolated antibody " refers to an antibody that is substantially free of other antibodies with different antigenic specificities (for example, the antibody that specifically binds the separation of HER2 is substantially free of antibodies that specifically bind the antigen except HER2). However, the antibody that specifically binds the separation of HER2 can have cross-binding reactivity with other antigens (such as HER2 proteins from other species, such as monkey HER2). Moreover, the isolated antibody can be substantially free of other cellular materials and / or chemical substances. In some embodiments, the recombinant polynucleotide of the coding polypeptide of the present invention or protein (such as anti-HER2 antibodies, anti-HER2 complementary bispecific antibodies) included in the carrier is considered to be isolated. Other examples of isolated polynucleotides are included in the recombinant polynucleotides included in the heterologous host cell or the polynucleotides of the purification (partially or substantially) in the solution.

[0082] As used herein, the term "engineering" includes any manipulation of the polypeptide or protein backbone, or post-translational modification of naturally occurring or recombinant proteins or polypeptides. Engineering includes mutations in the amino acid sequence, modifications to glycosylation or individual amino acid side chain groups, and combinations of these methods.

[0083] As used herein, the term "polypeptide" refers to a polymer of amino acids and their equivalents, rather than to a specific length of the product; therefore, "peptide" and "protein" are included within the definition of polypeptide. Also included within the definition of polypeptide is an "antibody" as defined herein.

[0084] Dual variable domain (DVD) immunoglobulin (DVD-Ig) is a tetravalent molecule that is constructed by inserting the heavy chain variable region (VH) and light chain variable region (VL) of another antibody into the N-terminus of the heavy chain and light chain of a normal IgG antibody, respectively, to produce a tetravalent molecule containing two different antigen-binding domains. Therefore, each half of a DVD-Ig contains a heavy chain polypeptide and a light chain polypeptide, which, after dimerization, form two independent antigen-binding domains, one of which is an Fv fragment and the other is a Fab fragment or IgG domain. DVD-Ig can be bispecific, i.e., it can bind to two different epitopes of an antigen, or bind to two different antigens.

[0085] As used herein, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted, leading to the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic material they carry.

[0086] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide or nucleic acid and / or vector has been introduced. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of generations. Progeny may not be identical in nucleic acid content to the parent cell and may contain mutations. The present invention includes mutant progeny screened or selected in the initially transformed cell that have the same function or biological activity.

[0087] As used herein, the terms "subject," "patient," or "individual" are used interchangeably and include, but are not limited to, mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, dogs, cats, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, sheep, or other non-human mammals; non-mammals, including, for example, non-mammalian vertebrates, such as birds (e.g., chickens, emus, or ducks) or fish; and non-mammalian invertebrates. In some embodiments, the subjects and pharmaceutical compositions of the uses or methods of the present invention are used to (prophylactically and / or therapeutically) treat non-human animals.

[0088] As used herein, "treating" or "treatment" of a disease or symptom refers to alleviating or alleviating a disease or symptom, reducing the rate of onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0089] The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration that is effective to prevent or improve symptoms associated with a disease or condition and / or reduce the severity of a disease or condition at a desired dose and for a desired period of time. The therapeutically effective amount of a formulation, antibody, or antigen-binding fragment thereof, ADC, or composition of the present invention can vary according to a variety of factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion or ADC to stimulate a desired response in the individual. A therapeutically effective amount can also be considered to be such that any toxic or deleterious effects of the formulation, antibody, or antigen-binding fragment thereof, ADC, or composition are outweighed by the therapeutically beneficial effects. The term "effective amount" refers to an amount of an active ingredient or agent sufficient to provide a clinical benefit to a subject, including but not limited to improving, alleviating, or alleviating a disease, condition, or associated symptoms, delaying or stopping disease progression.

[0090] As used herein, "low level of HER2", "low expression of HER2", "low HER2 expression", "low HER2 expression", or "low level of HER2 expression" refers to the expression of HER2 in paraffin-embedded tissue sections of tumor biopsies as determined by immunohistochemistry (IHC) (e.g. ) when ErbB2 / HER2 protein staining intensity was scored as IHC 1 + (faint / barely discernible membrane staining detected in more than 10% of tumor cells, cells stained only in a portion of their membrane), or when scored as IHC 2 + When weak to moderate whole-membrane staining is observed in more than 10% of tumor cells, the tumor is detected by fluorescence in situ hybridization (FISH) (e.g., Inform TM [Ventana], or PathVysion TM [Vysis]) further determined that the degree of HER2 gene amplification in tumor cells was negative (IHC 2 + / FISH - ) cancer cells / tumors, subjects or patients. Most HER2-low expressing tumors have HER2 expression levels significantly higher than those in normal tissues. The HER2 expression level in normal tissues can be determined by any method available to those skilled in the art.

[0091] "HER2 negative" or "HER2 expression negative" or "expressing limited levels of HER2" refers to cancer cells / tumors, subjects or patients that have low HER2 expression as described above and an IHC assay score of 0 (no staining observed or membranous staining present in less than 10% of tumor cells).

[0092] "HER2 positive" or "HER2 expression positive" or "HER2 overexpression" refers to an IHC score of IHC 3 + HER2 positive also includes an IHC assay score of IHC 2 + , cancer cells / tumors, subjects, or patients who score positive on both FISH tests (e.g., Subtractive Probe Technology Chromogenic In Situ Hybridization [SPoT-Light HER2 CISH] test, Inform Dual In Situ Hybridization [Inform HER2Dual ISH] test) + / FISH + ).

[0093] In this article, the "bystander effect" or "bystander killing effect" of ADC is mediated by free membrane-permeable small molecule toxins (e.g., anticancer agents), which are released by target molecule-positive (e.g., HER2-positive) cancer cells and passively diffuse into the tumor microenvironment, thereby killing neighboring cells, including neighboring cancer cells that do not express or low-express the target molecule (e.g., HER2-negative) and are insensitive to ADC.

[0094] As used herein, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" means that the referenced carriers, vehicles, diluents, excipients and / or salts are generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the subject.

[0095] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, or in one embodiment, a lower limit that is 10% less and an upper limit that is 10% greater, or in another embodiment, a lower limit that is 15% less and an upper limit that is 15% greater, or in another embodiment, a lower limit that is 20% less and an upper limit that is 20% greater.

[0096] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0097] As used herein, the terms "comprises" or "includes" or "contains" or "has" or "involving" are used interchangeably to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. In this document, when the terms "comprises" or "includes" or "contains" or "has" or "involving" are used, unless otherwise indicated, the context also encompasses consisting of the stated elements, integers or steps.

[0098] As used herein, the term "optionally" indicates that the object it modifies is present or absent, for example, "the kit optionally comprises at least one additional tumor therapeutic agent" means that the kit may or may not comprise at least one additional tumor therapeutic agent.

[0099] Reference herein to "some embodiments," "one embodiment," "a specific embodiment," or "a specific embodiment" or combinations thereof means that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0100] Unless the context clearly indicates otherwise, singular terms include plural referents and vice versa.

[0101] For the purpose of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because their disclosure predates the filing date of the present invention. All statements regarding the dates of these documents or the representation of the contents of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents.

[0102] Various aspects of the invention are described in more detail in the following sections.

[0103] 1. Anti-HER2 antibodies or antigen-binding fragments thereof

[0104] In one aspect, the present invention provides an isolated anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER2 antibody or the antigen-binding fragment thereof can specifically bind to the extracellular domain of HER2.

[0105] The anti-HER2 antibodies or antigen-binding fragments thereof of the present invention can specifically bind to D1, D3, and / or D4 of the HER2 extracellular region, preferably D1 or D3 of the HER2 extracellular region. The complementary anti-HER2 bispecific antibodies of the present invention can be constructed using the antigen-binding domains of two monospecific anti-HER2 antibodies, wherein the two monospecific antibodies can each bind to a different epitope in the HER2 extracellular region, and in an antigen competitive binding assay, the two monospecific antibodies do not exhibit competitive inhibition (i.e., the antigen-binding epitopes do not overlap).

[0106] In some embodiments, the antigen-binding domains of two monospecific anti-HER2 antibodies can be used to construct the anti-HER2 complementary bispecific antibodies of the present invention. The two monospecific antibodies can simultaneously bind to different epitopes in the extracellular region of HER2. Furthermore, the two monospecific antibodies can simultaneously bind to any two epitopes among D1, D3, and D4 in the extracellular region of HER2. Preferably, they can simultaneously bind to D1 and D3 in the extracellular region of HER2. For example, the antigen-binding domain of the first anti-HER2 antibody binds to D3 in the extracellular region of HER2, and the antigen-binding domain of the second anti-HER2 antibody binds to D1 in the extracellular region of HER2.

[0107] The anti-HER2 antibody or antigen-binding fragment thereof has strong binding activity to tumor cells expressing HER2, including HER2-high-expressing tumor cells (e.g., breast cancer cells SKBR-3, breast ductal cancer cells BT474, gastric cancer cells NCI-N87, and ovarian cancer cells SKOV-3), tumor cells expressing moderate levels of HER2 (e.g., breast cancer cells JIMT-1), HER2-low-expressing tumor cells (e.g., human bladder cancer cells RT-112, breast cancer cells ZR-75-1 and T47D), and / or tumor cells expressing limited levels of HER2 (e.g., breast cancer cells MCF-7). The binding affinity (K) of the anti-HER2 antibody or antigen-binding fragment thereof to HER2 is D ) value<5×10 -8 M, preferably <1×10 -8 M, <5×10 -9 M, or <1×10 -9 M, more preferably <1×10 -9 M. The anti-HER2 antibody or antigen-binding fragment thereof does not cross-bind with other members of the ErbB / HER family (including EGFR, HER3 and HER4).

[0108] The anti-HER2 antibody or its antigen-binding fragment does not affect the regulation of HER2 expressed on the cell surface and its downstream signal transduction pathways. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment does not induce, block, or inhibit the phosphorylation and / or dephosphorylation of tyrosine residues in the intracellular domain of HER2, and the tyrosine residue phosphorylation sites of the HER2 intracellular domain include but are not limited to Y877, Y1221 / 1222, and Y1248. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment does not induce, block, or inhibit HER2 dimerization (including HER2:HER4 dimerization and / or HER2:HER3 dimerization) and its downstream signal transduction pathways that are dependent on or independent of ligand induction, and the ligand includes NRG-1 or Heregulins.

[0109] The anti-HER2 antibodies of the present invention may also optionally include F(ab')2, Fab, Fab', Fv, scFv, scFv-Fc, single domain antibodies (sdAb), or have IgG types. The antibodies of the present invention may be murine antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and may be monoclonal antibodies, polyclonal antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies or antibody fragments, as long as the antibodies can specifically recognize epitopes of D1, D3 and / or D4 (preferably D1 and / or D3) of the HER2 extracellular region and have no effect on HER2 expressed on the surface of tumor cells and its signal transduction pathway. In some embodiments, the anti-HER2 antibodies are selected from mouse anti-human HER2 antibodies, humanized antibodies thereof, and optimized antibodies. In some embodiments, the anti-HER2 antibody comprises scFv, scFv-Fc, Fab fragment and / or has an IgG type. For example, an antibody that specifically binds to D3 of the extracellular region of HER2 can be in the form of scFv or scFv-Fc, and an antibody that specifically binds to D1 of the extracellular region of HER2 can be in the form of Fab or IgG, wherein Fab and scFv can be converted at will, and the conversion method is known in the art (see, for example, the method described in Zhou et al., Mol Cancer Ther 2012, 11: 1167-1476).

[0110] On the other hand, the present invention provides an anti-HER2 antibody or antigen-binding fragment thereof comprising the CDRs and / or variable regions of the antibodies shown in Table 2, which recognizes D3 of the HER2 extracellular region and does not induce, block, or inhibit the phosphorylation and / or dephosphorylation of tyrosine residues in the HER2 intracellular domain, and / or does not induce, block, or inhibit HER2 dimerization and its downstream signal transduction pathways that are dependent on or independent of NRG-1 induction. The present invention also provides an anti-HER2 antibody or antigen-binding fragment thereof that can recognize D1 of the HER2 extracellular region, which does not induce, block, or inhibit the phosphorylation and / or dephosphorylation of tyrosine residues in the HER2 intracellular domain, and / or does not induce, block, or inhibit HER2 dimerization and its downstream signal transduction pathways that are dependent on or independent of NRG-1 induction. Preferably, the antibody comprises the anti-HER2 antibody or antigen-binding fragment thereof comprising the CDRs and / or variable regions of the antibodies shown in Table 3. The anti-HER2 antibody or antigen-binding fragment thereof of the present invention further comprises CDRs, variable regions, or light and heavy chains of the anti-HER2 antibody or antigen-binding fragment thereof that recognizes the HER2 extracellular region D4.

[0111] The heavy chain variable region CDRs and light chain variable region CDRs of the anti-HER2 antibodies of the present invention are defined by the Kabat numbering system. However, as is known in the art, CDR regions may also be defined based on other numbering systems / methods for heavy chain / light chain variable region sequences, such as Chothia and IMGT, AbM, or Contact. CDR regions defined by other numbering systems / methods are also within the scope of protection of the present invention, as are CDR regions defined by the Kabat numbering system employed in the present invention.

[0112] Table 2. Amino acid sequence ID numbers of the CDR regions, heavy chain variable regions, and light chain variable regions of antibody mAb2117 and its humanized antibodies

[0113] Table 3. Amino acid sequence ID numbers of the CDR regions, heavy chain variable regions, and light chain variable regions of antibody mAb2126 and its humanized antibodies

[0114] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D3 of the extracellular region of HER2, and comprises the VH amino acid sequence shown in SEQ ID NO: 1 and / or the VL amino acid sequence shown in SEQ ID NO: 2.

[0115] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, and comprises a VH amino acid sequence as shown in SEQ ID NO: 3, 7, or 9 and / or a VL amino acid sequence as shown in SEQ ID NO: 4, 8, or 10. In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO: 3 and / or a VL amino acid sequence as shown in SEQ ID NO: 4. In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO: 7 and / or a VL amino acid sequence as shown in SEQ ID NO: 8. In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO: 9 and / or a VL amino acid sequence as shown in SEQ ID NO: 10.

[0116] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D4 of the extracellular region of HER2, and comprises the VH amino acid sequence shown in SEQ ID NO: 5 and / or the VL amino acid sequence shown in SEQ ID NO: 6.

[0117] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D3 of the extracellular region of HER2 of the present invention comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequences shown in SEQ ID NOs: 11, 14 and 32 or variants thereof, including humanized antibodies or any other variants described herein.

[0118] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D3 in the extracellular region of HER2 of the present invention further comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequences shown in SEQ ID NOs: 38, 46 and 49 or variants thereof, including humanized antibodies or any other variants described herein.

[0119] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49.

[0120] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 in the extracellular region of HER2 of the present invention comprises one or more CDRs of the VH amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequences shown in SEQ ID NOs: 98, 100 and 110, or variants thereof, one or more CDRs of the VH amino acid sequence shown in SEQ ID NO: 7, or the amino acid sequences shown in SEQ ID NOs: 205, 206 and 207, or variants thereof, and / or one or more CDRs of the VH amino acid sequence shown in SEQ ID NO: 9, or the amino acid sequences shown in SEQ ID NOs: 211, 212 and 213, or variants thereof, wherein the variants include humanized antibodies or any other variants described herein.

[0121] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 in the extracellular region of HER2 of the present invention comprises one or more CDRs in the VL amino acid sequence of SEQ ID NO:4, or the amino acid sequences as shown in SEQ ID NOs:114, 116 and 139, or variants thereof, one or more CDRs in the VL amino acid sequence of SEQ ID NO:8, or the amino acid sequences as shown in SEQ ID NOs:208, 209 and 210, or variants thereof, and / or one or more CDRs in the VL amino acid sequence of SEQ ID NO:10, or the amino acid sequences as shown in SEQ ID NOs:214, 215 and 216, or variants thereof, wherein the variants include humanized antibodies or any other variants described herein.

[0122] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100, and HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, and LCDR3 as shown in SEQ ID NO:139; or HCDR1 as shown in SEQ ID NO:205, HCDR2 as shown in SEQ ID NO:206, and HCDR3 as shown in SEQ ID NO:207, and LCDR1 as shown in SEQ ID NO:208, LCDR2 as shown in SEQ ID NO:209, and LCDR3 as shown in SEQ ID NO:210; or HCDR1 as shown in SEQ ID NO:211, HCDR2 as shown in SEQ ID NO:212, and HCDR3 as shown in SEQ ID NO:213, and LCDR1 as shown in SEQ ID NO:214, LCDR2 as shown in SEQ ID NO:215, and LCDR3 as shown in SEQ ID NO:216. LCDR2 shown in NO:215 and LCDR3 shown in SEQ ID NO:216.

[0123] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D4 of the extracellular region of HER2 of the present invention comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequences shown in SEQ ID NOs: 199, 200 and 201 or variants thereof, including humanized antibodies or any other variants described herein.

[0124] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D4 of the extracellular region of HER2 of the present invention comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequences shown in SEQ ID NOs: 202, 203 and 204 or variants thereof, including humanized antibodies or any other variants described herein.

[0125] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D4 of the extracellular region of HER2 of the present invention comprises HCDR1 as shown in SEQ ID NO: 199, HCDR2 as shown in SEQ ID NO: 200, and HCDR3 as shown in SEQ ID NO: 201, and LCDR1 as shown in SEQ ID NO: 202, LCDR2 as shown in SEQ ID NO: 203, and LCDR3 as shown in SEQ ID NO: 204.

[0126] On the other hand, the VH and / or VL of the anti-HER2 antibodies or antigen-binding fragments thereof of the present invention can be used as starting materials for engineering to prepare antibodies described herein that are more suitable for administration to humans. Antibodies can be engineered by mutating one or more amino acid residues within one or both variable regions (i.e., VH and / or VL), for example, by mutating one or more CDR regions and / or one or more FR framework regions.

[0127] In some embodiments, the anti-HER2 antibody variable region of the present invention is engineered by CDR grafting. The antibody interacts with the target antigen primarily through the amino acid residues of the six CDRs of the heavy chain variable region and the light chain variable region. Therefore, the amino acid sequence within the CDR region between each antibody is more diverse than the sequence (e.g., FR) outside the CDR region. Because the amino acid sequence of the CDR region is responsible for most of the interactions between the antibody and the antigen, recombinant antibodies can be expressed to mimic the properties of a specific naturally occurring antibody by constructing an expression vector that contains the CDR sequence from a specific naturally occurring antibody grafted onto the FR sequence of another antibody with different properties (see, e.g., Riechmann et al., Nature 1998, 332:323-327; Jones et al., Nature 1986, 321:522-525; Queen et al., Proc Natl Acad Sci USA 1989, 86:10029-10033; see also US5225539, US5530101, US5585089, US5693762, and US6180370).

[0128] The anti-HER2 antibodies of the present invention may also comprise different framework region sequences. Such framework region sequences can be obtained from public DNA databases or published references to germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "V Base" human germline sequence database (available at www.mrc-cpe.cam.ac.uk / vbase); and Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242; Tomlinson et al., J Mol Biol 1992, 227: 776-798; and Cox et al., Eur J Immunol 1994, 24: 827-836; the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the IMGT database, for example, the following heavy chain germline sequences found in human immunoglobulins can be obtained by IMGT accession number: IGHV3-23 (DP47; VH26; V3-23) or IGHV7-4. As another example, the following light chain germline sequences found in human immunoglobulins can be obtained by IMGT accession number: IGKV1-39 or IGKV1-39*01.

[0129] Antibody amino acid sequences can be compared against compiled protein sequence databases using one of the sequence similarity search methods known as Gapped BLAST, which is well known to those skilled in the art (Altschul et al., Nucleic Acids Res 1997, 25:3389-3402).

[0130] Preferred framework sequences for use in the anti-HER2 antibodies of the present invention are acceptor framework regions that are structurally similar (or highly homologous) to the framework sequences of the murine parental antibodies of the present invention. In some embodiments, the CDR1 region sequence, CDR2 region sequence, and CDR3 region sequence of the VH or VL can be transplanted into an acceptor framework region, wherein the acceptor framework region has a sequence that is identical to or has the highest homology to the germline immunoglobulin gene in which it is derived. In a specific embodiment, the present invention selects to transplant the CDR regions of VH shown in SEQ ID NO: 1 or 3 and VL shown in SEQ ID NO: 2 or 4 into the human IgG FR region to obtain a humanized antibody, wherein the humanized antibody not only maintains an antigen binding activity similar to that of the parent antibody comprising the VH shown in SEQ ID NO: 1 and the VL amino acid sequence shown in SEQ ID NO: 2, or the VH shown in SEQ ID NO: 3 and the VL amino acid sequence shown in SEQ ID NO: 4, but also does not affect the HER2 downstream signal transduction pathway, for example, it does not affect the phosphorylation of tyrosine residues in the HER2 intracellular domain (e.g., Y1248 phosphorylation) and / or ligand (e.g., NRG-1 or Heregulins)-induced HER2 dimerization and its mediated downstream signal transduction pathway.

[0131] In addition, the VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of other anti-HER2 antibodies that bind to HER2 can also be "mixed and matched" with the VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of the anti-HER2 antibodies of the present invention. Preferably, when the VH and VL chains (or the CDRs within these chains, or full-length heavy chain and full-length light chain sequences) are mixed and matched, the VH sequence from a specific VH / VL pair is replaced by a structurally similar VH sequence. Similarly, preferably, the VL sequence from a specific VH / VL pair is replaced by a structurally similar VL sequence. Similarly, the full-length heavy chain sequence from a specific full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the full-length light chain sequence from a specific full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.

[0132] Therefore, in one embodiment, the antibody of the present invention or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising the amino acid sequence listed in Table 2 or Table 3; and (b) a light chain variable region comprising the amino acid sequence listed in Table 2 or Table 3, or the VL of another anti-HER2 antibody, wherein the antibody specifically binds to D3 or D1 of the extracellular region of HER2.

[0133] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: (a) a heavy chain variable region comprising the amino acid sequence listed in Table 2 or Table 3, or VH of another anti-HER2 antibody, wherein the antibody specifically binds to D3 or D1 of the extracellular region of HER2; and (b) a light chain variable region comprising the amino acid sequence listed in Table 2 or Table 3.

[0134] In a specific embodiment, the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 52. The antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.

[0135] In a specific embodiment, the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 81. The antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.

[0136] In one embodiment, the humanized antibody of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 52, and a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 81. The antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.

[0137] In a specific embodiment, the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 143 or 170. The antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2.

[0138] In a specific embodiment, the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 171. The antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2.

[0139] In one embodiment, the humanized antibody of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 143 or 170, and a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 171. The antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular domain of HER2.

[0140] In some embodiments, the CDR sequence can be transplanted onto a framework region comprising one or more mutations compared to the germline sequence. For example, mutating amino acid residues within the framework region can maintain or enhance the antigen binding ability of the antibody (see, e.g., US5530101, US5585089, US5693762, and US6180370). In some embodiments, transplanting the CDRs of the parent antibody of the present invention onto an acceptor framework region having one or more mutations compared to the germline sequence can increase the degree of humanization of the anti-HER2 antibody of the present invention and / or improve antigen binding affinity.

[0141] In some specific embodiments, the FR region of the anti-HER2 humanized antibody that specifically binds to D3 of the extracellular region of HER2 of the present invention has one or more amino acid mutations to improve the antigen-binding affinity of the humanized antibody. For example, the FR region of the VH shown in SEQ ID NO: 52 is subjected to one or more amino acid mutations. Further, amino acid mutations can be made in HFR1, for example, an amino acid mutation is made at the serine residue at position 30 of HFR1 (corresponding to position 30 of the VH amino acid sequence shown in SEQ ID NO: 52); and / or the FR region of the VL shown in SEQ ID NO: 81 is subjected to one or more amino acid mutations. Further, one or more amino acid mutations can be made in LFR1, LFR2 and / or LFR3, specifically, one or more amino acid mutations are made at the aspartic acid residue at position 2, the methionine residue at position 4 of LFR1, the alanine residue at position 9 (corresponding to position 43 of the VL amino acid sequence shown in SEQ ID NO: 81), and the threonine residue at position 29 of LFR3 (corresponding to position 85 of the VL amino acid sequence shown in SEQ ID NO: 81).

[0142] In a specific embodiment, the anti-HER2 humanized antibody of the present invention that specifically binds to D3 of the extracellular region of HER2 has an amino acid mutation S30N in the HFR region relative to the VH amino acid sequence shown in SEQ ID NO: 52, and / or it has one or more amino acid mutations, including D2I, M4L, A43S, and T85V, in the LFR region relative to the VL amino acid sequence shown in SEQ ID NO: 81, which can significantly improve the antigen-binding affinity of the antibody.

[0143] In some specific embodiments, the FR region of the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 of the present invention has one or more amino acid mutations to improve the degree of humanization of the antibody of the present invention. For example, the FR region of the VH shown in SEQ ID NO: 52 is subjected to one or more amino acid mutations. Further, amino acid mutations can be made in HFR1 and / or HFR2, for example, amino acid mutations can be made in the tyrosine residue at position 29 of HFR1 and / or the alanine residue at position 14 of HFR2 (corresponding to position 49 of the amino acid sequence shown in SEQ ID NO: 52); and / or one or more amino acid mutations can be made in the FR region of the VL shown in SEQ ID NO: 81, and further, amino acid mutations can be made in LFR3, for example, amino acid mutations can be made in the phenylalanine residue at position 31 of LFR3 (corresponding to position 87 of the VL amino acid sequence shown in SEQ ID NO: 81). Specifically, the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 is mutated relative to the FR region of SEQ ID NO: 52. The VH amino acid sequence shown in NO:52 has the following amino acid mutations Y29F and / or A49S in the HFR, and / or it has an amino acid mutation F87Y in the LFR region relative to the VL amino acid sequence shown in SEQ ID NO:81, which can improve the degree of humanization of the antibody.

[0144] In some specific embodiments, the FR region of the anti-HER2 humanized antibody that specifically binds to D1 of the extracellular region of HER2 of the present invention has one or more amino acid mutations. For example, one or more amino acid mutations are made in the FR region of the VH shown in SEQ ID NO: 143, and one or more amino acid mutations can be made in HFR1 and / or HFR3 to improve the degree of humanization of the anti-HER2 antibody of the present invention. Preferably, one or more amino acid mutations are performed at the serine residue at position 9, the valine residue at position 18 of HFR1, and / or the valine residue at position 3 (corresponding to position 68 of the VH amino acid sequence set forth in SEQ ID NO: 143), the phenylalanine residue at position 4 (corresponding to position 69 of the VH amino acid sequence set forth in SEQ ID NO: 143), the leucine residue at position 6 (corresponding to position 71 of the VH amino acid sequence set forth in SEQ ID NO: 143), the valine residue at position 10 (corresponding to position 75 of the VH amino acid sequence set forth in SEQ ID NO: 143), the isoleucine residue at position 17 (corresponding to position 82 of the VH amino acid sequence set forth in SEQ ID NO: 143), and the phenylalanine residue at position 29 (corresponding to position 91 of the VH amino acid sequence set forth in SEQ ID NO: 143) of HFR3. One or more amino acid mutations may be made in the FR region of the VL shown in SEQ ID NO: 171 to improve the degree of humanization of the anti-HER2 antibody of the present invention. For example, an amino acid mutation may be made at the asparagine residue at position 15 of LFR2 (corresponding to position 49 of the VL amino acid sequence shown in SEQ ID NO: 171).

[0145] In a specific embodiment, the anti-HER2 humanized antibody D1 that specifically binds to the extracellular region of HER2 of the present invention has one or more of the following amino acid mutations in the HFR region relative to the VH amino acid sequence set forth in SEQ ID NO: 143: S9G, V18L, V68T or V68S, F69I, L71V or L71R, V75K or V75T, I82L or I82M, F91Y. In some embodiments, the humanized antibody D1 that specifically binds to the extracellular region of HER2 comprises one or more of the following amino acid mutations in the HFR region relative to the VH amino acid sequence set forth in SEQ ID NO: 143: V68T, F69I, L71R, V75K, and F91Y, which can significantly improve the degree of humanization of the antibody.

[0146] In some embodiments, the present invention performs amino acid mutations in the CDR regions of the VH and / or VL of the humanized antibody to improve one or more properties of the antibody, for example, to increase the degree of humanization, improve antigen binding affinity, increase expression, enhance stability (e.g., reduce potential aspartic acid isomerization and / or asparagine deamidation risks). Site-directed mutagenesis or PCR-induced mutagenesis can be performed, and the effect of the mutation on the functional properties of the antibody can be evaluated by in vitro or in vivo detection methods known in the art. The mutation can be an amino acid substitution, addition or deletion, preferably an amino acid substitution. In particular, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues are mutated in the heavy chain CDR or light chain CDR regions.

[0147] In some specific embodiments, the anti-HER2 humanized antibody of the present invention that specifically binds to D3 in the extracellular region of HER2 has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations in the heavy chain CDR region or the light chain CDR region.

[0148] In a specific embodiment, one or more amino acid mutations are made in the CDR region of the VH shown in SEQ ID NO: 52. Furthermore, one, two, three, four, or five amino acid mutations may be made in HCDR1, HCDR2 and / or HCDR3. For example, one or more amino acid mutations may be made at the serine residue at position 1 of HCDR1 (corresponding to position 31 of the VH set forth in SEQ ID NO: 52), at the glycine residue at position 5 (corresponding to position 53 of the VH set forth in SEQ ID NO: 52), at the serine residue at position 7 (corresponding to position 55 of the VH set forth in SEQ ID NO: 52), at the threonine residue at position 9 (corresponding to position 57 of the VH set forth in SEQ ID NO: 52), at the proline residue at position 12 (corresponding to position 60 of the VH set forth in SEQ ID NO: 52), at the aspartic acid residue at position 13 (corresponding to position 61 of the VH set forth in SEQ ID NO: 52), at the serine residue at position 14 (corresponding to position 62 of the VH set forth in SEQ ID NO: 52), and / or at the alanine residue at position 3 (corresponding to position 97 of the VH set forth in SEQ ID NO: 52) of HCDR2.

[0149] In one embodiment, one or more amino acid mutations are made to HCDR1, HCDR2 and / or HCDR3 in the VH set forth in SEQ ID NO: 52, for example, amino acid mutations are made to the serine residue at position 7 (corresponding to position 55 of the VH set forth in SEQ ID NO: 52), the threonine residue at position 9 (corresponding to position 57 of the VH set forth in SEQ ID NO: 52), the proline residue at position 12 (corresponding to position 60 of the VH set forth in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 of the VH set forth in SEQ ID NO: 52), the serine residue at position 14 (corresponding to position 62 of the VH set forth in SEQ ID NO: 52), and / or the alanine residue at position 3 (corresponding to position 97 of the VH set forth in SEQ ID NO: 52) of HCDR2 of the VH set forth in SEQ ID NO: 52.

[0150] In a specific embodiment, one or more amino acid mutations are made to the CDR region in the VL of SEQ ID NO: 81. Furthermore, one, two, or three amino acid mutations may be made in LCDR1, LCDR3, and / or LCDR3. For example, one or more amino acid mutations may be made at the valine residue at position 7 (corresponding to position 27c of the VL of SEQ ID NO: 81), the histidine residue at position 8 (corresponding to position 27d of the VL of SEQ ID NO: 81), the glycine residue at position 11 (corresponding to position 29 of the VL of SEQ ID NO: 85) in LCDR1, at the phenylalanine residue at position 6 (corresponding to position 55 of the VL of SEQ ID NO: 81) in LCDR2, and / or at the serine residue at position 1 (corresponding to position 89 of the VL of SEQ ID NO: 81), and the tyrosine residue at position 8 (corresponding to position 96 of the VL of SEQ ID NO: 81) in LCDR3.

[0151] In one embodiment, the present invention performs multiple amino acid mutations on the CDR regions of the VH and VL of the above-mentioned anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 to improve the degree of humanization of the antibody of the present invention, increase the expression level, and / or improve the stability (for example, reduce the potential risk of aspartic acid isomerization). Specifically, one or more amino acid mutations were made to the serine residue at position 7 of HCDR2 (corresponding to position 55 of VH shown in SEQ ID NO: 52), the threonine residue at position 9 (corresponding to position 57 of VH shown in SEQ ID NO: 52), the proline residue at position 12 (corresponding to position 60 of VH shown in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 of VH shown in SEQ ID NO: 52), the serine residue at position 14 (corresponding to position 62 of VH shown in SEQ ID NO: 52), and the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH shown in SEQ ID NO: 52), and the glycine residue at position 11 of LCDR1 of VL shown in SEQ ID NO: 81 (corresponding to position 29 of VL shown in SEQ ID NO: 81) was mutated. Preferably, amino acid mutations are performed on any one or two of the proline residue at position 12 of HCDR2 of the VH shown in SEQ ID NO: 52 (corresponding to position 60 of the VH shown in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 of the VH shown in SEQ ID NO: 52), and the serine residue at position 14 (corresponding to position 62 of the VH shown in SEQ ID NO: 52), and on the alanine residue at position 3 of HCDR3 (corresponding to position 97 of the VH shown in SEQ ID NO: 52), and on the glycine residue at position 11 of LCDR1 of the VL shown in SEQ ID NO: 81 (corresponding to position 29 of the VL shown in SEQ ID NO: 81).

[0152] In some embodiments, the anti-HER2 humanized antibody of the present invention that specifically binds to D1 in the extracellular region of HER2 has no more than 1, 2, 3, 4, or 5 amino acid mutations in the heavy chain CDR region or the light chain CDR region.

[0153] In a specific embodiment, one or more amino acid mutations are made in the CDR region of the VH shown in SEQ ID NO: 143. Furthermore, one, two, three, four, or five amino acid mutations may be made in HCDR1, HCDR2 and / or HCDR3. For example, one or more amino acid mutations may be made at the serine residue at position 3 of HCDR1 (corresponding to position 33 of the VH set forth in SEQ ID NO: 143), at the glutamic acid residue at position 5 (corresponding to position 53 of the VH set forth in SEQ ID NO: 143), at the glutamic acid residue at position 8 (corresponding to position 56 of the VH set forth in SEQ ID NO: 143), at the aspartic acid residue at position 14 (corresponding to position 62 of the VH set forth in SEQ ID NO: 143), at the phenylalanine residue at position 15 (corresponding to position 63 of the VH set forth in SEQ ID NO: 143) of HCDR2, and / or at the arginine residue at position 3 (corresponding to position 97 of the VH set forth in SEQ ID NO: 143), at the tyrosine residue at position 4 (corresponding to position 98 of the VH set forth in SEQ ID NO: 143), at the aspartic acid residue at position 5 (corresponding to position 99 of the VH set forth in SEQ ID NO: 143) of HCDR3.

[0154] In a specific embodiment, one or more amino acid mutations are made in the CDR region of the VL set forth in SEQ ID NO: 171. For example, one or more amino acid mutations can be made at the lysine residue at position 1 of LCDR1 (corresponding to position 24 of the VL set forth in SEQ ID NO: 171), at the serine residue at position 1 (corresponding to position 50 of the VL set forth in SEQ ID NO: 171), at the tyrosine residue at position 4 (corresponding to position 53 of the VL set forth in SEQ ID NO: 171), at the tyrosine residue at position 6 (corresponding to position 55 of the VL set forth in SEQ ID NO: 171), and / or at the histidine residue at position 3 of LCDR3 (corresponding to position 91 of the VL set forth in SEQ ID NO: 171).

[0155] In one embodiment, the present invention performs multiple amino acid mutations on the CDR regions of VH and VL of the above-mentioned anti-HER2 humanized antibody that specifically binds to D1 in the extracellular region of HER2 to improve the degree of humanization of the antibody of the present invention, enhance antigen binding affinity, increase expression level, and / or improve stability (for example, reducing the risk of potential asparagine deamidation). Specifically, multiple amino acid mutations were performed on the glutamic acid residue at position 5 of HCDR2 of the VH shown in SEQ ID NO: 143 (corresponding to position 53 of the VH shown in SEQ ID NO: 143), and the lysine residue at position 1 of LCDR1 of the VL shown in SEQ ID NO: 171 (corresponding to position 24 of the VL shown in SEQ ID NO: 171), the serine residue at position 1 of LCDR2 (corresponding to position 50 of the VL shown in SEQ ID NO: 171), the tyrosine residue at position 4 (corresponding to position 53 of the VL shown in SEQ ID NO: 171), the tyrosine residue at position 6 (corresponding to position 55 of the VL shown in SEQ ID NO: 171) and the histidine residue at position 3 of LCDR3 (corresponding to position 91 of the VL shown in SEQ ID NO: 171).

[0156] In some embodiments, multiple amino acid mutations can also be made to the CDR and framework regions of the variable regions of the humanized antibody to further improve one or more properties of the antibody (e.g., increasing the degree of humanization, increasing antigen binding affinity, enhancing stability, and increasing expression). In some specific embodiments, multiple amino acid mutations are performed on one or two CDR regions and one HFR region of the VH shown in SEQ ID NO: 143, and / or the three CDR regions of the VL shown in SEQ ID NO: 171 of the humanized antibody that specifically binds to the extracellular region of HER2, preferably, the glutamic acid residue at position 5 of HCDR2 of the VH shown in SEQ ID NO: 143 (corresponding to position 53 of the VH shown in SEQ ID NO: 143), the valine residue at position 3 of HFR3 (corresponding to position 68 of the amino acid sequence of the VH shown in SEQ ID NO: 143), the phenylalanine residue at position 4 (corresponding to position 69 of the amino acid sequence of the VH shown in SEQ ID NO: 143), the leucine residue at position 6 (corresponding to position 71 of the amino acid sequence of the VH shown in SEQ ID NO: 143), the valine residue at position 10 (corresponding to position 75 of the amino acid sequence of the VH shown in SEQ ID NO: 143), the phenylalanine residue at position 29 (corresponding to position 30 of the amino acid sequence of the VH shown in SEQ ID NO: 171), the leucine residue at position 10 (corresponding to position 76 of the amino acid sequence of the VH shown in SEQ ID NO: 171), the phenylalanine residue at position 29 (corresponding to position 31 of the amino acid sequence of the VH shown in SEQ ID NO: 171), the glutamic acid residue at position 5 of HCDR2 of the VH shown in SEQ ID NO: 143 (corresponding to position 53 of the VH shown in SEQ ID NO: 143), the valine residue at position 3 of HFR3 (correspond NO: 143), and / or multiple amino acid mutations are performed on the 1st lysine residue of LCDR1 (corresponding to the 24th VL position of SEQ ID NO: 171), the 1st serine residue of LCDR2 (corresponding to the 50th VL position of SEQ ID NO: 171), the 4th tyrosine residue (corresponding to the 53rd VL position of SEQ ID NO: 171), the 6th tyrosine residue (corresponding to the 55th VL position of SEQ ID NO: 171), and the 3rd histidine residue of LCDR3 (corresponding to the 91st VL position of SEQ ID NO: 171), thereby not only making the humanized antibody of the present invention have a higher degree of humanization, but also significantly improving the binding affinity to HER2 (K D Value is about 8×10 -9 ~9×10 -10 M, preferably 5×10 -9 ~9×10 -10 M), while also reducing the potential risk of asparagine deamidation and increasing expression levels.

[0157] In other embodiments, the present invention provides an isolated anti-HER2 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to D3 of the extracellular region of HER2, and comprises (1) a HCDR1 having an amino acid sequence of X1YGMS (X1=S, N and D; i.e., the sequence shown in SEQ ID NO: 217), preferably, X1=S; (2) an amino acid sequence of SISGX2GX3YX4KYX5X6X7VKG (X2=G or S; X3=S or N; X4=T or A; X5=P, A, G or V; X6=D, G, E, P, Q or R; X7=S, K or N; i.e., the sequence shown in SEQ ID NO: 217). (i.e., the sequence shown in SEQ ID NO: 218), preferably, X2=G, X3=S, X4=T, X5=P or V, X6=D, P or E, X7=S or K; (3) a HCDR3 with the amino acid sequence DYX8GFFDV (X8=A, I, N, R, S, or V; i.e., the sequence shown in SEQ ID NO: 219), preferably, X8=V; (4) an amino acid sequence RSSQSLX9X 10 SNX 11 NTYLH(X9=V or L;X 10 =H or S; X 11 =G, A, I, S, R or T; i.e., the sequence shown in SEQ ID NO: 220), preferably, X9 =V or L, X 10 =H,X 11 =R; (5) amino acid sequence is KVSNRX 12 S(X 12 =F, D or P; i.e., the sequence shown in SEQ ID NO: 221), preferably, X 12 =F; (6) amino acid sequence is X 13 QSTHVPX 14 T(X 13 =S or Q; X 14 =Y or W; i.e., the sequence shown in SEQ ID NO: 222), preferably, X 13 =S,X 14 =Y, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The antibody or antigen-binding fragment thereof has a high degree of humanization, a high expression level, and eliminates the risk of aspartic acid isomerization.

[0158] In some specific embodiments, the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to D3 of the extracellular region of HER2 comprises: HCDR1 as shown in SEQ ID NO: 11, 12, or 13; HCDR2 as shown in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31; and HCDR3 as shown in SEQ ID NO: 32, 33, 34, 35, 36, or 37; and LCDR1 as shown in SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 45; LCDR2 as shown in SEQ ID NO: 46, 47, or 48; and LCDR3 as shown in SEQ ID NO: 49, 50, or 51.

[0159] In a specific embodiment, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention specifically binds to D3 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively:

[0160] (1) HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20, HCDR3 as set forth in SEQ ID NO: 32; and LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 46, LCDR3 as set forth in SEQ ID NO: 49; or

[0161] (2) HCDR1 as set forth in SEQ ID NO: 12 or 13, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 32, and LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 46, LCDR3 as set forth in SEQ ID NO: 49; or

[0162] (3) HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 14, HCDR3 as shown in SEQ ID NO: 32, and LCDR1 as shown in SEQ ID NO: 38, LCDR2 as shown in SEQ ID NO: 47 or 48, LCDR3 as shown in SEQ ID NO: 49; or

[0163] (4) HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 32, and LCDR1 as set forth in SEQ ID NO: 39, 40, 41, 42, 43, 44, or 45, LCDR2 as set forth in SEQ ID NO: 46, or LCDR3 as set forth in SEQ ID NO: 49; or

[0164] (5) HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 32, and LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 46, LCDR3 as set forth in SEQ ID NO: 50 or 51; or

[0165] (6) HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 33, 34, 35, 36, or 37; and LCDR1 as set forth in SEQ ID NO: 38, LCDR2 as set forth in SEQ ID NO: 46, LCDR3 as set forth in SEQ ID NO: 49; or

[0166] (7) HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 14, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, HCDR3 as set forth in SEQ ID NO: 37; and LCDR1 as set forth in SEQ ID NO: 42, LCDR2 as set forth in SEQ ID NO: 46, LCDR3 as set forth in SEQ ID NO: 49; or

[0167] (8) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49.

[0168] In a specific embodiment, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention specifically binds to D3 of the extracellular region of HER2 and comprises: a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 14, 17, 19, 21, 23, 24, 27, 28, 29, 30, or 31, a HCDR3 as shown in SEQ ID NO: 32 or 37, and a LCDR1 as shown in SEQ ID NO: 38 or 42, a LCDR2 as shown in SEQ ID NO: 46, a LCDR3 as shown in SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. Preferably, the antibody or antigen-binding fragment thereof comprises: a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 14, 17, 19, 24, 29, 30, or 31, a HCDR3 as shown in SEQ ID NO: 32 or 37, and a LCDR1 as shown in SEQ ID NO: 38 or 42, a LCDR2 as shown in SEQ ID NO: 46, a LCDR3 as shown in SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. More preferably, the antibody or antigen-binding fragment thereof comprises: a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 24, 29, 30, or 31, and a HCDR3 as shown in SEQ ID NO: 37, and a LCDR1 as shown in SEQ ID NO: 42, a LCDR2 as shown in SEQ ID NO: 46, and a LCDR3 as shown in SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0169] In other embodiments, the present invention provides an isolated anti-HER2 monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2, and comprises (1) an amino acid sequence of DYX 15 MH(X 15 =S or A; i.e., the sequence shown in SEQ ID NO: 223) of HCDR1, preferably, X 15 =S; (2) amino acid sequence is WINTX 16 TGX 17 PTYADX 18 X 19 KG(X 16 =E, N, G, Y, or I; X 17 =E, D or S; X 18 =D, K or N; X 19 =F or V; i.e., the HCDR2 of the sequence shown in SEQ ID NO: 224), preferably, X 16 =G,X 17 =E,X 18 =D,X 19 =F; (3) amino acid sequence is VGX 20 X 21 X 22 YAMDY(X 20 =R or Y; X 21 =Y or G; X 22 =D or S; i.e., the sequence shown in SEQ ID NO: 225), preferably, X 20 =R,X 21 =Y,X 22 =D; (4) amino acid sequence is X 23 ASQDVYTAVA(X 23 =K or R; i.e., the sequence shown in SEQ ID NO: 226) LCDR1, preferably, X 23 =R; (5) amino acid sequence is X 24 ASX 25 RX 26 T(X 24 =S, A, D, E, K, L, Q, R, W or Y; X 25 =Y, D, E, K, N, Q, S, or T; X 26 =Y, A, E, P or Q; i.e., the sequence shown in SEQ ID NO: 227), preferably, X 24 =S, A, L or Y, X 25 =S,X 26 =P; (6) amino acid sequence is QQX 27 YSTPPT(X27 =H, S, A or Y; i.e., the sequence shown in SEQ ID NO: 228), preferably, X 27 = Y, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The antibody or antigen-binding fragment thereof not only has high affinity for HER2 (e.g., K D Value <1×10 -8 M, or <5×10 -9 M, or <1×10 -9 M, preferably K D Value <5×10 -9 M, or <1×10 -9 M), and has a higher degree of humanization and stability.

[0170] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 comprises: a HCDR1 as shown in SEQ ID NO: 98 or 99; a HCDR2 as shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109; a HCDR3 as shown in SEQ ID NO: 110, 111, 112, or 113; and a LCDR1 as shown in SEQ ID NO: 114 or 115; a LCDR2 as shown in SEQ ID NO: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138; LCDR3 shown in NO:139, 140, 141, or 142.

[0171] In a specific embodiment, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention specifically binds to D1 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively:

[0172] (1) a HCDR1 as set forth in SEQ ID NO:98, a HCDR2 as set forth in SEQ ID NO:100, a HCDR3 as set forth in SEQ ID NO:110, a LCDR1 as set forth in SEQ ID NO:114, a LCDR2 as set forth in SEQ ID NO:116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, or 128, or a LCDR3 as set forth in SEQ ID NO:139; or

[0173] (2) HCDR1 as set forth in SEQ ID NO:98, HCDR2 as set forth in SEQ ID NO:100, HCDR3 as set forth in SEQ ID NO:110, LCDR1 as set forth in SEQ ID NO:114, LCDR2 as set forth in SEQ ID NO:116, LCDR3 as set forth in SEQ ID NO:140, 141, or 142; or

[0174] (3) a HCDR1 as set forth in SEQ ID NO: 98, a HCDR2 as set forth in SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, or 109, a HCDR3 as set forth in SEQ ID NO: 110, a LCDR1 as set forth in SEQ ID NO: 114, a LCDR2 as set forth in SEQ ID NO: 116, or a LCDR3 as set forth in SEQ ID NO: 139; or

[0175] (4) HCDR1 as set forth in SEQ ID NO: 98, HCDR2 as set forth in SEQ ID NO: 100, HCDR3 as set forth in SEQ ID NO: 111, 112, or 113, LCDR1 as set forth in SEQ ID NO: 114, LCDR2 as set forth in SEQ ID NO: 116, or LCDR3 as set forth in SEQ ID NO: 139; or

[0176] (5) HCDR1 as set forth in SEQ ID NO:98, HCDR2 as set forth in SEQ ID NO:100, HCDR3 as set forth in SEQ ID NO:110, LCDR1 as set forth in SEQ ID NO:115, LCDR2 as set forth in SEQ ID NO:116, LCDR3 as set forth in SEQ ID NO:139; or

[0177] (6) HCDR1 as set forth in SEQ ID NO:99, HCDR2 as set forth in SEQ ID NO:100, HCDR3 as set forth in SEQ ID NO:110, LCDR1 as set forth in SEQ ID NO:114, LCDR2 as set forth in SEQ ID NO:116, LCDR3 as set forth in SEQ ID NO:139; or

[0178] (7) HCDR1 as set forth in SEQ ID NO:98, HCDR2 as set forth in SEQ ID NO:102, HCDR3 as set forth in SEQ ID NO:110, LCDR1 as set forth in SEQ ID NO:115, LCDR2 as set forth in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, LCDR3 as set forth in SEQ ID NO:142.

[0179] In a specific embodiment, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively: HCDR1 as shown in SEQ ID NO: 98, HCDR2 as shown in SEQ ID NO: 100 or 102, HCDR3 as shown in SEQ ID NO: 110, LCDR1 as shown in SEQ ID NO: 114 or 115, LCDR2 as shown in NO:116, 123, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and LCDR3 as shown in SEQ ID NO:139 or 142. Preferably, the antibody or antigen-binding fragment thereof comprises: a HCDR1 as set forth in SEQ ID NO: 98, a HCDR2 as set forth in SEQ ID NO: 102, a HCDR3 as set forth in SEQ ID NO: 110, a LCDR1 as set forth in SEQ ID NO: 115, a LCDR2 as set forth in SEQ ID NO: 129, 133, 136, or 138, a LCDR3 as set forth in SEQ ID NO: 142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The binding affinity (K) of the antibody or antigen-binding fragment thereof to HER2 is D ) value<5×10 -9 M, or <1×10 -9 M, preferably <1×10 -9M. More preferably, the antibody or antigen-binding fragment thereof comprises: a HCDR1 as set forth in SEQ ID NO: 98, a HCDR2 as set forth in SEQ ID NO: 102, and a HCDR3 as set forth in SEQ ID NO: 110, and a LCDR1 as set forth in SEQ ID NO: 115, a LCDR2 as set forth in SEQ ID NO: 133, and a LCDR3 as set forth in SEQ ID NO: 142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0180] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3.

[0181] In some specific embodiments, the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to D3 of the extracellular region of HER2, comprising: a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, and a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97 has a light chain variable region VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical.

[0182] In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention can specifically bind to D3 of the extracellular region of HER2, which comprises a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 52 and the VL shown in SEQ ID NO: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, respectively; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 52 and the VL shown in SEQ ID NO: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 72, 73, 74, 75, 76, 77, 78, 79, or 80 and the VH set forth in SEQ ID NO: 81; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 and the VL set forth in SEQ ID NO: 81; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 and the VL set forth in SEQ ID NO: The VL shown in NO:92 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to D3 of the extracellular region of HER2 comprises: an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in any one of SEQ ID NOs: 52, 57, 62, 68, 73, 78, 79 and 80, and the VL shown in SEQ ID NO: 81 or 92. Preferably, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH of SEQ ID NO: 73, 78, 79, or 80 and the VL of SEQ ID NO: 92, respectively. More preferably, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH of SEQ ID NO: 80 and the VL of SEQ ID NO: 92, respectively.

[0184] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, comprising: a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170, and a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: NO:171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 has a light chain variable region VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence shown in FIG.

[0185] In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises a heavy chain variable region comprising said HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising said LCDR1, LCDR2 and LCDR3, which comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 143 and the VL shown in SEQ ID NO: 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188, respectively; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 143 and the VL shown in SEQ ID NO: 171, 172, 173, 174, 175, : 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 170 and the VL of SEQ ID NO: 171 having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 169 and the VL of SEQ ID NO: 170, respectively. The VL shown in NO:189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH of any one of SEQ ID NOs: 143, 148, 156, 158, 160, 161, 165, and 169, and the VL of any one of SEQ ID NOs: 171, 172, 180, 184, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. Preferably, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH of SEQ ID NO: 169 and the VL of SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. More preferably, the VH of SEQ ID NO: 169 and the VL of SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. The VL of SEQ ID NO: 189, 193, 196, or 198 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Further preferably, the antibody or antigen-binding fragment thereof of the present invention comprises: the VH of SEQ ID NO: 169, and the VL of SEQ ID NO: 193.

[0187] The anti-HER2 humanized antibody or antigen-binding fragment thereof of the present invention has strong binding activity with cells expressing HER2, has no effect on the HER2-mediated signal transduction pathway, does not undergo cross-binding reactions with other members of the human ErbB / HER family (including EGFR, HER3 and HER4), and has a high degree of humanization and stability.

[0188] 2. Anti-HER2 complementary bispecific antibodies

[0189] On the one hand, the present invention provides an anti-HER2 complementary bispecific antibody, the complementary bispecific antibody comprising two antigen-binding domains capable of simultaneously and non-competitively binding to the HER2 extracellular region, wherein the first antigen-binding domain and the second antigen-binding domain each specifically bind to a different epitope of the HER2 extracellular region. Further, the complementary bispecific antibody can simultaneously bind to any two epitopes of D1, D3, and D4 in the HER2 extracellular region, preferably, can simultaneously bind to D1 and D3 in the HER2 extracellular region, for example, wherein the first antigen-binding domain specifically binds to D3 in the HER2 extracellular region, and the second antigen-binding domain specifically binds to D1 in the HER2 extracellular region, and the binding epitopes of the first and second antigen-binding domains are different from the antigen-binding epitopes of trastuzumab or pertuzumab.

[0190] The anti-HER2 complementary bispecific antibodies of the present invention have the following functional properties:

[0191] (i) The anti-HER2 complementary bispecific antibody is a tetravalent molecule that can simultaneously bind to two different epitopes in the extracellular region of HER2, thereby cross-linking HER2 on the cell surface to form antigen-antibody cross-linked aggregates or clusters. Obviously, the efficiency and size of the aggregate or cluster formation are subject to the abundance of the target antigen on the cell surface, and the formation of the cluster will trigger the cell's endocytosis reaction, and the rate and intensity of the endocytosis reaction are positively correlated with the size of the cluster. Specifically, compared with the corresponding monospecific anti-HER2 antibody or antigen-binding fragment thereof and trastuzumab, the complementary bispecific antibody has a significantly enhanced internalization efficiency, with an endocytosis rate of over 80% in HER2-overexpressing tumor cells and an endocytosis rate of over 60% in HER2-low-expressing tumor cells.

[0192] (ii) Unlike the corresponding monospecific anti-HER2 antibodies or antigen-binding fragments thereof and trastuzumab, which are primarily transported to the cell surface (recycling) after internalization, the complementary bispecific antibody forms clusters with HER2 on the cell surface and undergoes internalization, with lysosomal trafficking being the dominant intracellular transport pathway. Therefore, the clusters formed by the complementary bispecific antibody and HER2 can be efficiently transported into lysosomes for degradation.

[0193] (iii) The complementary bispecific antibody can effectively trigger the degradation of HER2 on the surface of tumor cells, thereby significantly inhibiting the proliferation of HER2-overexpressing tumor cells.

[0194] (iv) The complementary bispecific antibody does not affect the regulation of HER2 and its mediated downstream signal transduction pathways, including not inducing, blocking, or inhibiting ligand (e.g., NRG-1)-induced HER2 dimerization and activation of its downstream signal transduction pathways. Therefore, the anti-HER2 complementary bispecific antibody does not affect the normal biological function and regulation of HER2 and its mediated downstream signal transduction pathways in normal tissues or cells (e.g., cardiomyocytes).

[0195] (v) The anti-HER2 complementary bispecific antibody has high stability and expression level, as well as a high monomer content (about 98% or more).

[0196] The complementary bispecific antibody formats include, but are not limited to, bispecific formats based on scFv or Diabody (e.g., scFv-scFv or scFv-Fab or Fab-scFv), IgG-scFv fusion proteins, DVD-Ig, Quadroma, Knob-into-hole, Common Light Chain, CrossMab, CrossFab, SEEDbody, Leucine Zipper, Duobody, IgG1 / IgG2, Dual-acting Fab (DAF)-IgG and Mab 2 Bispecific format (see, e.g., Klein et al., mAbs 2012, 4:653-663 and references cited therein). The exemplary anti-HER2 complementary bispecific antibody of the present invention is in the form of a DVD-Ig, which not only has high binding activity against HER2-expressing tumor cells, but also exhibits a high cellular internalization rate. In addition, the stability of the complementary bispecific antibody of the present invention can be ensured (e.g., a high monomer content of about 98% or more).

[0197] The anti-HER2 complementary bispecific antibody has strong binding activity with cells expressing HER2 and has no cross-binding activity with other members of the human ErbB / HER family (including EGFR, HER3 and HER4).

[0198] In tumor cells expressing HER2, the anti-HER2 complementary bispecific antibodies exhibit significantly enhanced internalization efficiency. In some embodiments, the complementary bispecific antibodies exhibit significantly enhanced internalization efficiency compared to the corresponding monospecific anti-HER2 antibodies or antigen-binding fragments thereof and trastuzumab in HER2 overexpression (IHC 3 + , or IHC 2 + / FISH +In some embodiments, the complementary bispecific antibody has a higher endocytosis rate in tumor cells with low HER2 expression (IHC 2 + / FISH - , or IHC 1 + ) tumor cells has an endocytosis rate of up to 60%.

[0199] The anti-HER2 complementary bispecific antibody can effectively promote the degradation of HER2 in tumor cells. In some embodiments, the complementary bispecific antibody can induce the degradation of HER2 in HER2-overexpressing tumor cells (e.g., BT474 cells) by Western-blot detection, while the monospecific anti-HER2 antibody or its antigen-binding fragment corresponding to the complementary bispecific antibody and Trastuzumab cannot induce the degradation of HER2 in the tumor cells. Further, the anti-HER2 complementary bispecific antibody can effectively reduce the expression abundance of HER2 on the surface of tumor cells, so the anti-HER2 complementary bispecific antibody can significantly inhibit the proliferation of HER2-overexpressing tumor cells. In some embodiments, the anti-HER2 complementary bispecific antibody has a significant inhibitory effect on the proliferation of HER2-overexpressing tumor cells (e.g., BT474 cells), and its inhibitory activity is comparable to that of Trastuzumab, while the monospecific anti-HER2 antibody or its antigen-binding fragment corresponding to the complementary bispecific antibody cannot inhibit the proliferation of tumor cells.

[0200] The anti-HER2 complementary bispecific antibody has no effect on the HER2-mediated signal transduction pathway, and maintains the characteristic that the corresponding monospecific anti-HER2 antibody or its antigen-binding fragment does not affect the biological function of HER2, and therefore does not adversely affect the normal biological function of HER2 in normal tissues or cells (e.g., cardiomyocytes). In some embodiments, the anti-HER2 complementary bispecific antibody does not induce, block, or inhibit the phosphorylation and / or dephosphorylation of AKT in the downstream signaling pathway induced by ligand (e.g., NRG-1)-induced HER2 dimerization and its mediation. Thus, unlike existing therapeutic agents targeting HER2 (e.g., therapeutic agents based on Trastuzumab and / or Pertuzumab), the anti-HER2 complementary bispecific antibody has an extremely low risk of potentially inducing cardiac toxic side effects.

[0201] On the other hand, the first and second antigen-binding domains of the complementary anti-HER2 bispecific antibody of the present invention can be derived from any two monospecific anti-HER2 antibodies or antigen-binding fragments thereof of the present invention that do not compete with each other for binding to HER2, or can also be derived from currently known anti-HER2 antibodies or antigen-binding fragments thereof.

[0202] Exemplary anti-HER2 complementary bispecific antibodies of the present invention can be constructed based on D3 that specifically binds to the extracellular region of HER2 as shown in Table 2 and the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to D1 that specifically binds to the extracellular region of HER2 as shown in Table 3. Exemplary anti-HER2 complementary bispecific antibodies of the present invention comprise two different antigen-binding domains, wherein the first antigen-binding domain comprises at least one CDR region and / or any variable region of the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D3 that specifically binds to the extracellular region of HER2 as shown in Table 2, and the second antigen-binding domain comprises at least one CDR region and / or any variable region of the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 that specifically binds to the extracellular region of HER2 as shown in Table 3.

[0203] In some embodiments, the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and / or light chain variable region CDRs, and the heavy chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two or three of the heavy chain variable region CDRs listed in Table 2, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 200%, 201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209 ... 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence; the light chain variable region CDRs of the first antigen binding domain comprise the amino acid sequence of any one, two or three of the light chain variable region CDRs listed in Table 2, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LCDR1, LCDR2 and LCDR3, respectively. The heavy chain variable region CDRs of the second antigen-binding domain comprise the amino acid sequences of any one, two or three of the heavy chain variable region CDRs listed in Table 3, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with HCDR1, HCDR2 and HCDR3, respectively. , or 100% identical amino acid sequence to LCDR1, LCDR2, and LCDR3; the light chain variable region CDRs of the second antigen-binding domain comprise the amino acid sequence of any one, two, or three CDR regions listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to LCDR1, LCDR2, and LCDR3, respectively.

[0204] In some embodiments, the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2 and comprises: a HCDR1 as set forth in SEQ ID NO:11, a HCDR2 as set forth in SEQ ID NO:21, 23, 24, 27, 28, 29, 30, or 31, a HCDR3 as set forth in SEQ ID NO:37, and a LCDR1 as set forth in SEQ ID NO:42, a LCDR2 as set forth in SEQ ID NO:46, and a LCDR3 as set forth in SEQ ID NO:49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0205] In a specific embodiment, the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2 and comprises: a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 24, 29, 30, or 31, a HCDR3 as shown in SEQ ID NO: 37, and a LCDR1 as shown in SEQ ID NO: 42, a LCDR2 as shown in SEQ ID NO: 46, and a LCDR3 as shown in SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0206] In some embodiments, the second antigen-binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2 and comprises: HCDR1 as shown in SEQ ID NO: 98, HCDR2 as shown in SEQ ID NO: 102, HCDR3 as shown in SEQ ID NO: 110, and LCDR1 as shown in SEQ ID NO: 115, LCDR2 as shown in SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and LCDR3 as shown in SEQ ID NO: 139. The LCDR3 shown in NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0207] In a specific embodiment, the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2 and comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133, and LCDR3 as shown in SEQ ID NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0208] In some embodiments, the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody further comprises a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 and / or a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the heavy chain variable region of the first antigen-binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the light chain variable region of the first antigen-binding domain comprises an amino acid sequence of any one of the VL amino acid sequences listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. The heavy chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 3, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 3, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity thereto; The light chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VL amino acid sequences listed in Table 3, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the light chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VL amino acid sequences listed in Table 3, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0209] In some specific embodiments, the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2 and comprises: a VH as shown in SEQ ID NO: 70, 72, 73, 76, 77, 78, 79, or 80, and a VL as shown in SEQ ID NO: 92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively.

[0210] In a specific embodiment, the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2, and comprises: a VH as shown in SEQ ID NO: 73, 78, 79, or 80, and a VL as shown in SEQ ID NO: 92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively.

[0211] In some specific embodiments, the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2 and comprises: a VH as shown in SEQ ID NO: 169, and a VL as shown in SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively.

[0212] In a specific embodiment, the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2, and comprises: VH as shown in SEQ ID NO: 169, and VL as shown in SEQ ID NO: 193, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively.

[0213] In some embodiments, the anti-HER2 complementary bispecific antibody comprises a first and a second antigen binding domain, wherein the first antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as set forth in SEQ ID NO: 37, and LCDR1 as set forth in SEQ ID NO: 42, LCDR2 as set forth in SEQ ID NO: 46, and an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively. NO:49; and the second antigen-binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as set forth in SEQ ID NO:98, HCDR2 as set forth in SEQ ID NO:102, HCDR3 as set forth in SEQ ID NO:110, and LCDR1 as set forth in SEQ ID NO:115, LCDR2 as set forth in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and LCDR3 as set forth in SEQ ID NO:142.

[0214] In a specific embodiment, the anti-HER2 complementary bispecific antibody comprises a first and a second antigen binding domain, wherein the first antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 24, 29, 30, or 31, HCDR3 as set forth in SEQ ID NO: 37, and LCDR1 as set forth in SEQ ID NO: 42, LCDR2 as set forth in SEQ ID NO: 46, and HCDR3 as set forth in SEQ ID NO: 50. NO:49, and the second antigen-binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133, and LCDR3 as shown in SEQ ID NO:142.

[0215] In some embodiments, the anti-HER2 complementary bispecific antibody comprises a first and a second antigen-binding domain, the first antigen-binding domain comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH and the light chain variable region VL comprising the VH amino acid sequence of SEQ ID NO: 70, 72, 73, 76, 77, 78, 79, or 80, and the VL amino acid sequence of SEQ ID NO: 92, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively, and the second antigen-binding domain comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH and the light chain variable region VL comprising the VH amino acid sequence of SEQ ID NO: 169 and the VL amino acid sequence of SEQ ID NO: 170. The VL amino acid sequence shown in NO:193, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.

[0216] In a specific embodiment, the anti-HER2 complementary bispecific antibody comprises a first and a second antigen-binding domain, the first antigen-binding domain comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH and the light chain variable region VL comprising the VH amino acid sequence as shown in SEQ ID NO: 73, 78, 79, or 80, and the VL amino acid sequence as shown in SEQ ID NO: 92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively, and the second antigen-binding domain comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH and the light chain variable region VL comprising the VH amino acid sequence as shown in SEQ ID NO: 169 and the VL amino acid sequence as shown in SEQ ID NO: 170. The VL amino acid sequence shown in NO:193, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.

[0217] Exemplary anti-HER2 complementary bispecific antibodies of the present invention can also be constructed based on the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to D1 and D4 of the HER2 extracellular region described in the present invention. Exemplary anti-HER2 complementary bispecific antibodies of the present invention comprise first and second antigen-binding domains, and the first and second antigen-binding domains are capable of simultaneously and non-competitively binding to HER2, wherein the first antigen-binding domain comprises the VH amino acid sequence of SEQ ID NO:7 and the VL amino acid sequence of SEQ ID NO:8, or the first antigen-binding domain comprises the VH amino acid sequence of SEQ ID NO:9 and the VL amino acid sequence of SEQ ID NO:10, or the first antigen-binding domain comprises at least one CDR region and / or any variable region of the anti-HER2 antibodies or antigen-binding fragments thereof shown in Table 3, and is capable of specifically binding to D1 of the HER2 extracellular region; and the second antigen-binding domain comprises the VH amino acid sequence of SEQ ID NO:5 and the VL amino acid sequence of SEQ ID NO:6, and is capable of specifically binding to D4 of the HER2 extracellular region.

[0218] In some embodiments, the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and / or light chain variable region CDRs, and the heavy chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs of the VH amino acid sequence of SEQ ID NO: 7, or the amino acid sequences of SEQ ID NOs: 205, 206, and 207, or variants thereof, including humanized antibodies or any other variants; the light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs of the VL amino acid sequence of SEQ ID NO: 8, or the amino acid sequences of SEQ ID NOs: 208, 209, and 210, or variants thereof, including humanized antibodies or any other variants; or, the heavy chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs of the VH amino acid sequence of SEQ ID NO: 9, or the amino acid sequences of SEQ ID NOs: 106, 107, and 108. NOs:211, 212 and 213 or variants thereof, including humanized antibodies or any other variants; the light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs of the VL amino acid sequence of SEQ ID NO:10 or the amino acid sequences of SEQ ID NOs:214, 215 and 216 or variants thereof, including humanized antibodies or any other variants; or the heavy chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two or three CDR regions of the heavy chain variable region CDRs listed in Table 3, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more similarity thereto. the light chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two, or three of the light chain variable region CDRs listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the heavy chain variable region CDRs of the second antigen-binding domain comprise one, two, or three of the VH amino acid sequence set forth in SEQ ID NO: 5, or the amino acid sequences set forth in SEQ ID NOs: 199, 200, and 201, or variants thereof, including humanized antibodies or any other variants thereof;The light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs of the VL amino acid sequence set forth in SEQ ID NO: 6, or the amino acid sequences set forth in SEQ ID NOs: 202, 203, and 204, or variants thereof, including humanized antibodies or any other variants.

[0219] Exemplary anti-HER2 complementary bispecific antibodies of the present invention can also be constructed based on the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to D3 and D4 of the HER2 extracellular region described in the present invention. Exemplary anti-HER2 complementary bispecific antibodies of the present invention comprise a first and a second antigen-binding domain, and the first and second antigen-binding domains are capable of simultaneously binding to HER2 in a non-competitive manner, wherein the first antigen-binding domain comprises at least one CDR region and / or any variable region of the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to D3 of the HER2 extracellular region as shown in Table 2; and the second antigen-binding domain comprises the VH amino acid sequence shown in SEQ ID NO: 5 and the VL amino acid sequence shown in SEQ ID NO: 6, and is capable of specifically binding to D4 of the HER2 extracellular region.

[0220] In some embodiments, the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and / or light chain variable region CDRs, and the heavy chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two, or three of the heavy chain variable region CDRs listed in Table 2, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 200%, 201%. The light chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two, or three of the light chain variable region CDRs listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the heavy chain variable region CDRs of the second antigen-binding domain comprise SEQ The light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs in the VH amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequences shown in SEQ ID NOs: 199, 200 and 201, or variants thereof, including humanized antibodies or any other variants; the light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs in the VL amino acid sequence shown in SEQ ID NO: 6, or the amino acid sequences shown in SEQ ID NOs: 202, 203 and 204, or variants thereof, including humanized antibodies or any other variants.

[0221] On the other hand, the exemplary anti-HER2 complementary bispecific antibodies of the present invention can be constructed in the form of DVD-Ig (see US7612181), and the first or second antigen binding domain of the anti-HER2 complementary bispecific antibody can be an Fv form / domain, or a Fab or IgG form / domain, that is, if the first antigen binding domain is Fv, then the second antigen binding domain is Fab or IgG; if the second antigen binding domain is Fv, then the first antigen binding domain is Fab or IgG. The Fv domain is fused to or operably connected to the Fab or IgG domain via a linker sequence, and the linker not only has low immunogenicity but also ensures the stability of the complementary bispecific antibody, preferably a flexible peptide as a linker.

[0222] As used herein, the term "linker" refers to a linker that connects two compounds, such as two polypeptide molecules (including but not limited to unmodified or modified amino acids or amino acid sequences). A linker can be composed of one or more linker molecules, or can include a linker molecule and at least one spacer molecule intended to separate the linker molecule and the compound by a specific distance.

[0223] The term "operably linked" refers to the linkage of amino acid sequences, peptides or proteins with different functional properties, such as the linkage of an Fv domain to a Fab or IgG domain via a linker sequence as described herein.

[0224] In some embodiments, the first antigen-binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2 and is an Fv domain, and the first antigen-binding domain comprises the heavy chain variable region CDRs and light chain variable region CDRs, and / or the heavy chain variable region VH and light chain variable region VL of the anti-HER2 antibodies or antigen-binding fragments thereof shown in Table 2; the second antigen-binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2 and is a Fab domain or an IgG domain. The second antigen-binding domain comprises the heavy chain variable region CDRs and light chain variable region CDRs, and / or the heavy chain variable region VH and light chain variable region VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 3; the C-terminus of the VH domain in the Fv domain is fused to or operably linked to the N-terminus of the VH domain in the Fab or IgG domain via a linker sequence, and the C-terminus of the VL domain in the Fv domain is fused to or operably linked to the N-terminus of the VL domain in the Fab or IgG domain via a linker sequence.

[0225] In some specific embodiments, the linker sequence includes different copy numbers of GGGGS (G4S) sequences, for example, 1, 2, 3, 4, or 5 copies. The copy number of the linker G4S connecting the VH domain in the Fv domain of the anti-HER2 complementary bispecific antibody to the VH domain in the Fab or IgG domain of the complementary bispecific antibody can be the same as or different from the copy number of the linker G4S connecting the VL domain in the Fv domain of the complementary bispecific antibody to the VL domain in the Fab or IgG domain of the complementary bispecific antibody. Preferably, the copy number of the linker G4S connecting the VH domain between the Fv domain and the Fab or IgG domain is different from the copy number of the linker G4S connecting the VL domain between the two. The copy number of the linker connecting the VH domains of the two is preferably 1, and the copy number of the linker connecting the VL domains of the two is preferably 3. This can effectively ensure the structural stability of the anti-HER2 complementary bispecific antibody of the present invention and improve the endocytosis rate in HER2-expressing tumor cells.

[0226] In a specific embodiment, the anti-HER2 complementary bispecific antibody comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises the VH and VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 2, and the second antigen-binding domain comprises the VH and VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 3, the first antigen-binding domain is an Fv domain, the second antigen-binding domain is a Fab or IgG domain, and the C-terminus of the VH domain in the Fv domain is connected by a linker (e.g., (G4S) n , n is an integer greater than 0, such as 1-3; preferably (G4S) 1 is connected to the N-terminus of the VH domain in the Fab or IgG domain, and the C-terminus of the VL domain in the Fv domain is connected via a linker (e.g. (G4S) n , n is an integer from 1 to 3; preferably (G4S)3) is connected to the N-terminus of the VL domain in the Fab or IgG domain.

[0227] On the other hand, the anti-HER2 complementary bispecific antibody of the present invention may further comprise a constant region comprising an antibody heavy chain constant region and a light chain constant region. The heavy chain constant region of the present invention comprises the natural and mutant protein forms of the human IgG heavy chain constant region Fc region, and also comprises a truncated form of a polypeptide containing a hinge region that promotes dimer formation. In some embodiments, the Fc region comprises antibody CH2 and CH3 domains. Fusion proteins comprising the Fc portion (and the oligomers formed therefrom) provide the advantage of being easily purified by affinity chromatography by protein A (Protein A) or protein G (Protein G), as well as extending serum half-life. The preferred Fc region is derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In this article, the position of the specific amino acid residues in the Fc region is determined according to the EU numbering system.

[0228] One function of the Fc region of an antibody is to produce "effector functions" with the immune system when the antibody binds to its target molecule, including the production of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by the binding of Fc to Fc receptors (FcRs) on the surface of immune cells (Raghavan et al., Annu Rev Cell Dev Biol 1996, 12: 181-220; Ghetie et al., Annu Rev Immunol 2000, 18: 739-766; Ravetch et al., Annu Rev Immunol 2001, 19: 275-290), including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, NK cells and T cells. CDC is mediated by binding of the Fc to proteins of the complement system, such as CIq (Ward et al., Ther Immunol 1995, 2:77-94).

[0229] In some embodiments, the anti-HER2 complementary bispecific antibodies of the present invention comprise an engineered IgG Fc region to reduce Fc-mediated effector function. Exemplary antibodies with reduced effector function include Fc regions containing the following amino acid mutations:

[0230] N297A or N297Q (IgG1)

[0231] S267E / L328F (IgG1)

[0232] L234A / L235A (IgG1)

[0233] L234F / L235E / P331S (IgG1)

[0234] C220S / C226S / C229S / P238S(IgG1)

[0235] C226S / C229S / E233P / L234V / L235A(IgG1)

[0236] V234A / G237A (IgG2)

[0237] H268Q / V309L / A330S / A331S(IgG2)

[0238] L235A / G237A / E318A (IgG4)

[0239] A preferred engineered Fc region is a human IgG1 Fc with L234F / L235E / P331S (EU numbering system) amino acid substitutions, which can reduce binding of the Fc region to one or more FcγRs and C1q (Oganesyan et al., Acta Crystallogr D Biol Crystallogr 2008, 64:700-704; US5624821; US6194551). The FcγR protein family includes FcγRI (also known as CD64), including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (also known as CD32), including isoforms FcγRIIa, FcγRIIb and FcγRIIc; and FcγRIII (also known as CD16), including isoforms FcγRIIIa and FcγRIIIb (Jefferis et al., Immunol Lett 2002, 82:57-65). Among them, FcγRI, FcγRIIa, FcγRIIc and FcγRIIIa can induce ADCC, endocytosis, phagocytosis and / or cytokine release. Binding properties include, but are not limited to, binding specificity, binding affinity (K D ), and the dissociation and association rates (k dis and k a ), those skilled in the art can analyze whether the engineered Fc region has altered ADCC and / or CDC activity by any one or more binding properties. In some embodiments, the heavy chain constant region and light chain constant region in the anti-HER2 complementary bispecific antibodies of the present invention can be a heavy chain constant region from human IgG1 or IgG4 (which can be native or mutated, such as the wild-type human IgG1 constant region shown in SEQ ID NO: 231), and a human Kappa (κ) chain or Lambda (λ) constant region.

[0240] In some specific embodiments, the heavy chain constant region of the anti-HER2 complementary bispecific antibody comprises a human IgG1 Fc (as shown in SEQ ID NO: 232) with L234F / L235E / P331S (EU numbering system) amino acid substitutions, which has reduced binding affinity for one or more FcγRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIa) and C1q. In some embodiments, the introduction of the L234F / L235E / P331S mutations into the heavy chain constant region of the anti-HER2 complementary bispecific antibody results in the complementary bispecific antibody not substantially binding to FcγRI, FcγRIIa(167H), FcγRIIb, FcγRIIIa(176V), and / or FcγRIIIa(176F). In some embodiments, the introduction of the L234F / L235E / P331S mutations into the heavy chain constant region of the complementary anti-HER2 bispecific antibody renders the complementary bispecific antibody substantially non-binding to C1q. In some embodiments, the introduction of the L234F / L235E / P331S mutations into the heavy chain constant region of the complementary anti-HER2 bispecific antibody does not affect the binding affinity of the complementary bispecific antibody to FcRn. In some embodiments, the introduction of the L234F / L235E / P331S mutations into the heavy chain constant region of the complementary anti-HER2 bispecific antibody significantly reduces the ADCC activity of the complementary bispecific antibody. In one embodiment, the ADCC activity of the complementary bispecific antibody is significantly reduced or undetectable compared to an antibody without the amino acid mutations in the Fc region. The reduction or abolition of ADCC activity may be due to a significant reduction in the binding affinity of the complementary bispecific antibody to FcγR.

[0241] In some specific embodiments, the anti-HER2 complementary bispecific antibody comprises a light chain constant region, and the light chain constant region is selected from the kappa constant region (as shown in SEQ ID NO: 233).

[0242] In one aspect, the anti-HER2 complementary bispecific antibody of the present invention comprises four polypeptide chains, wherein two polypeptide chains comprise VH1-L1-VH2-C-(Fc)n, VH1 represents the heavy chain variable region of the first antigen binding domain of D3 that specifically binds to the extracellular region of HER2, L1 represents a linker, VH2 represents the heavy chain variable region of the second antigen binding domain of D1 that specifically binds to the extracellular region of HER2, C represents the heavy chain constant region CH1, Fc represents the heavy chain constant region Fc domain, and n is 0 or 1; and the other two polypeptide chains comprise VL1-L2-VL2-CL, wherein VL1 represents the light chain variable region of the first antigen binding domain of D3 that specifically binds to the extracellular region of HER2, L2 represents a linker, VL2 represents the light chain variable region of the second antigen binding domain of D1 that specifically binds to the extracellular region of HER2, and CL is an IgG light chain constant region.

[0243] In some embodiments, the VH1 comprises the HCDR1 amino acid sequence of SEQ ID NO: 11, the HCDR2 amino acid sequence of SEQ ID NO: 24, 29, 30, or 31, and the HCDR3 amino acid sequence of SEQ ID NO: 37, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, and HCDR3, respectively; the L1 comprises the linker amino acid sequence of SEQ ID NO: 229; the VH2 comprises the HCDR1 amino acid sequence of SEQ ID NO: 98, the HCDR2 amino acid sequence of SEQ ID NO: 102, and the HCDR3 amino acid sequence of SEQ ID NO: 37. The invention also provides an embodiment of the present invention wherein the present invention comprises an amino acid sequence of SEQ ID NO: 110, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, and HCDR3, respectively; and an Fc domain comprising a human IgG1 Fc having L234F / L235E / P331S (EU numbering system) amino acid substitutions, the amino acid sequence of which is shown in SEQ ID NO: 232.

[0244] In some embodiments, the VH1 comprises the VH amino acid sequence of SEQ ID NO:73, 78, 79, or 80, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and the VH2 comprises the VH amino acid sequence of SEQ ID NO:169, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0245] In some embodiments, the VL1 comprises the LCDR1 amino acid sequence of SEQ ID NO:42, the LCDR2 amino acid sequence of SEQ ID NO:46, and the LCDR3 amino acid sequence of SEQ ID NO:49, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said LCDR1, LCDR2, and LCDR3, respectively; L2 comprises the linker amino acid sequence of SEQ ID NO:230; VL2 comprises the LCDR1 amino acid sequence of SEQ ID NO:115, the LCDR2 amino acid sequence of SEQ ID NO:133, and the LCDR3 amino acid sequence of SEQ ID NO:144. The LCDR3 amino acid sequence shown in NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said LCDR1, LCDR2, and LCDR3, respectively; CL is selected from a human kappa constant region or a human lambda constant region, preferably a human kappa constant region (such as the amino acid sequence shown in SEQ ID NO:233).

[0246] In some embodiments, the VL1 comprises the VL amino acid sequence of SEQ ID NO:92, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and the VL2 comprises the VL amino acid sequence of SEQ ID NO:193, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0247] The complementary anti-HER2 bispecific antibodies of the present invention can specifically and simultaneously bind to two different epitopes in the extracellular region of HER2, thereby effectively cross-linking HER2 on the cell surface to form antigen-antibody cross-linked aggregates or clusters, thereby inducing cellular endocytosis and promoting the internalized clusters to be transported to lysosomes and degraded within the lysosomes. As a result, the complementary bispecific antibodies can significantly downregulate the expression of HER2 on the cell surface, reduce HER2 dimerization, and thus inhibit the proliferation of tumor cells that overexpress HER2. Furthermore, the complementary anti-HER2 bispecific antibodies of the present invention do not interfere with the HER2-mediated signaling pathway, and therefore do not interfere with the normal biological functions of HER2 in normal tissues / cells.

[0248] 3. Anti-HER2 complementary bispecific antibody-drug conjugate (ADC)

[0249] The present invention further provides an anti-HER2 complementary bispecific ADC, comprising a small molecule toxin compound coupled to an anti-HER2 complementary bispecific antibody of the present invention via a cleavable linker, which has one or more of the following functional properties:

[0250] (i) The anti-HER2 complementary bispecific ADC is a tetravalent molecule that can specifically bind to two non-overlapping epitopes of HER2 on the surface of tumor cells, thereby effectively cross-linking HER2 and forming cross-linked aggregates or clusters on the tumor cell membrane. On the one hand, the formation of clusters causes it to be quickly and efficiently internalized by cells, and the intracellular transport pathway after internalization is changed from the usual recycling (Recycling) to lysosomal transport (Lysosomal Trafficking), which significantly improves the efficiency of the ADC transport into the lysosome, thereby being able to release more small molecule toxin compounds into the cytoplasm after degradation in the lysosome. Therefore, the complementary bispecific ADC has stronger targeted cytotoxicity, which is manifested in tumor cells overexpressing HER2 (IHC 3 + , or IHC 2 + / FISH + ) has stronger killing activity than existing HER2-targeted drugs (e.g., Trastuzumab, Pertuzumab, T-DM1, DS-8201) and is effective in HER2-low expressing tumor cells (IHC 2 + / FISH - , or IHC 1 + ) can also exert a direct killing effect, potentially expanding the scope of HER2-targeted therapy indications. Furthermore, the released small molecule toxin compound is hydrophobic and cell membrane permeable, allowing it to passively diffuse into the tumor microenvironment and exert a bystander effect, killing adjacent tumor cells that express lower levels of HER2 or no HER2. In summary, compared to conventional ADCs, this complementary bispecific ADC has a broader spectrum of tumor cell killing activity and is less likely to develop drug resistance.

[0251] (ii) The anti-HER2 complementary bispecific ADC has a killing effect on tumors that have become resistant or relapsed to trastuzumab, pertuzumab, T-DM1, and DS-8201, thereby solving the drug resistance problem of existing HER2 targeted drugs.

[0252] (iii) The anti-HER2 complementary bispecific ADC does not affect the dimerization of HER2 and the regulation of its mediated signal transduction pathways, and therefore does not interfere with the normal biological function of HER2 in cardiomyocytes, thereby greatly reducing the possibility of causing cardiac toxic side effects.

[0253] (iv) The anti-HER2 complementary bispecific ADC has significantly reduced Fc receptor binding affinity and thus has extremely low Fc receptor-mediated safety risks.

[0254] The ADC can be represented by formula (I):

[0255] Ab-(LD)p(Ⅰ)

[0256] wherein Ab represents the anti-HER2 complementary bispecific antibody of the present invention;

[0257] D represents a small molecule toxin compound (Drug);

[0258] L represents a cleavable linker that couples Ab to D; and

[0259] p represents the copy number of (LD) coupled to the Ab, ranging from 2 to 8.

[0260] The Ab in the ADC is an anti-HER2 complementary bispecific antibody of the present invention, which comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises the VH and VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 2, can specifically bind to D3 of the HER2 extracellular region, and is an Fv domain; the second antigen-binding domain comprises the VH and VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 3, can specifically bind to D1 of the HER2 extracellular region, and is a Fab domain or an IgG domain.

[0261] In some embodiments, the first antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise: (i) HCDR1 as set forth in SEQ ID NO: 11; (ii) HCDR2 as set forth in SEQ ID NO: 24, 29, 30, or 31; (iii) HCDR3 as set forth in SEQ ID NO: 37; (iv) LCDR1 as set forth in SEQ ID NO: 42; (v) LCDR2 as set forth in SEQ ID NO: 46; and (vi) LCDR3 as set forth in SEQ ID NO: 49; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0262] In some embodiments, the second antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise: (i) HCDR1 as set forth in SEQ ID NO:98; (ii) HCDR2 as set forth in SEQ ID NO:102; (iii) HCDR3 as set forth in SEQ ID NO:110; (iv) LCDR1 as set forth in SEQ ID NO:115; (v) LCDR2 as set forth in SEQ ID NO:133; and (vi) LCDR3 as set forth in SEQ ID NO:142; or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0263] In some embodiments, the first antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a VH as set forth in SEQ ID NO: 73, 78, 79, or 80, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a VL as set forth in SEQ ID NO: 92, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0264] In some embodiments, the second antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a VH as set forth in SEQ ID NO: 169, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a VL as set forth in SEQ ID NO: 193, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0265] D in the ADC is a small molecule toxin compound that, when not conjugated to the anti-HER2 complementary bispecific antibody of the present invention, exhibits cytotoxic or cytostatic effects on both tumor cells and normal cells. However, after conjugation to form an ADC, the small molecule toxin compound only exhibits cytotoxic and / or bystander killing effects after the ADC is internalized and transported into the lysosomes of target cells expressing HER2 and dissociates from the ADC through enzymatic action, hydrolysis, oxidation, or any other mechanism. Such small molecule toxin compounds include cytotoxins and chemotherapeutic drugs.

[0266] In some embodiments, the cytotoxins include microtubule inhibitors and DNA damaging agents, the microtubule inhibitors include Eribulin, Auristatins derivatives (e.g., MMAE, MMAF, MMAD), Tubulysins, Cryptomycins and Maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4), the DNA damaging agents include topoisomerase inhibitors (e.g., camptothecin derivatives SN-38, Exatecan and DXd), pyrrolobenzodiazepines (PBD), and calicheamicin and its derivatives (e.g., N-acetylcalicheamicin [CMC]), Duocarmycin, and the preferred small molecule toxin compound is Eribulin.

[0267] The molecular structure of Eribulin is shown in formula (II):

[0268] The term "eribulin" refers to a synthetic analog of halichondria B, a macrocyclic compound isolated from the marine sponge Halichondria okadais. Eribulin acts as a microtubule dynamics inhibitor by binding to tubulin, inhibiting the formation of the mitotic spindle and causing cell division cycle arrest at the G2 / M phase. The structures of exemplary eribulin or its analogs and methods for their synthesis are described in WO1999065894, WO2004034990, ZL201910197071.8, and / or ZL201910509222.9, the disclosures of which are incorporated herein by reference.

[0269] In some embodiments, the small molecule toxin compound is eribulin, which has the characteristics of a wide clinical therapeutic window and low off-target cytotoxicity.

[0270] In some embodiments, the chemotherapeutic drug can be a natural or synthetic compound, including but not limited to alkyl chemotherapeutic agents and other compounds with alkylated forms (e.g., nitrogen mustards, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, dacarbazine), antimetabolites (e.g., folic acid, purine or pyrimidine antagonists), mitotic inhibitors (e.g., vinca alkaloids and derivatives of podophyllotoxin), cytotoxic antibiotics (e.g., anthracycline antibiotics including daunorubicin and doxorubicin, as well as actinomycin, bleomycin).

[0271] The cleavable linker is stable outside the tumor cells expressing HER2, so that the ADC of the present invention maintains structural stability in vitro or in the blood circulation, and does not cause systemic toxicity (off-target effect) due to the non-targeted or random release of small molecule toxin compounds. At the same time, after endocytosis into tumor cells expressing HER2, it can quickly enter the lysosome for degradation and release the small molecule toxin compound to kill tumor cells. The cleavable linker refers to any linker comprising a cleavable portion. As used herein, the term "cleavable portion" refers to any chemical bond that can be cleaved, for example, as well known in the art, including but not limited to, a bond that is unstable to acid, a bond that is unstable to proteases / peptidases, a bond that is unstable to light, a bond that is unstable to esterases, and a disulfide bond. The linker comprising a cleavable portion can allow the small molecule toxin compound to dissociate from the ADC via cleavage at a specific site in the linker.

[0272] In some embodiments, the cleavable linker comprises a cleavable peptide portion, which is cleaved by an intracellular protease (e.g., an endosomal protease, a lysosomal protease, or a tumor-associated protease) to release a small molecule toxin compound from the ADC to kill tumor cells. In some embodiments, the cleavable peptide portion comprises an amino acid unit that can be cleaved by a lysosomal cysteine ​​cathepsin (e.g., cathepsin B, C, F, H, K, L, O, S, V, X, or W). The amino acid unit may comprise naturally occurring amino acid residues and / or non-naturally occurring amino acid analogs, such as citrulline (Citrulline, abbreviated as Cit). In some embodiments, the amino acid unit comprises a dipeptide, a tripeptide, or a tetrapeptide, for example, a Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG) amino acid sequence that can be cleaved by cathepsin B, preferably an amino acid unit comprising VC and GGFG.

[0273] In some embodiments, the cleavable linker may comprise at least one spacer that couples the small molecule toxin compound (D) to the anti-HER2 complementary bispecific antibody (Ab) of the present invention, wherein the spacer comprises a spacer conjugated to the antibody, and / or a second spacer conjugated to the small molecule compound.

[0274] In some embodiments, the spacer conjugated to the antibody can be hydrophilic to increase the hydrophilicity of the ADC and improve its stability, reduce ADC product aggregation, and reduce immunogenicity. Exemplary spacers include one or more polyethylene glycols (PEGs), such as 1, 2, 3, 4, 5, or 6 PEGs, preferably 2 PEGs. In some embodiments, the spacer is attached to the antibody via maleimide (Mal), wherein the spacer attached to the antibody via Mal is also referred to herein as a "Mal-spacer." In some embodiments, the Mal-spacer comprises one or more PEG moieties (e.g., 2 PEGs).

[0275] In some embodiments, the second spacer conjugated to the small molecule compound is used to attach the cleavable portion of the cleavable linker (e.g., a cleavable peptide) to the small molecule compound. In some embodiments, the second spacer conjugated to the small molecule compound has the property of self-immolation, which facilitates the complete release of the small molecule compound within the target cell. That is, the small molecule compound released within the target cell does not carry the conjugated spacer portion or other modifying groups, so that the released small molecule compound is not affected by the presence of modifying groups and its anti-tumor activity. In some embodiments, the self-immolation spacer comprises a p-aminobenzyl unit, p-aminobenzyl alcohol (PABOH) is conjugated to the cleavable portion of the cleavable linker (e.g., an amino acid unit of the cleavable peptide) via an amide bond, and a carbamate group, a methylcarbamate group, or a carbonate group is formed between the PABOH and the small molecule compound (Hamann et al., Expert Opinion on Therapeutic Patents 2005, 15:1087-1103). Furthermore, the self-immolative fragmentation spacer comprises a p-aminobenzylcarbonyl (PAB) group represented by formula (III), and the self-immolative fragmentation of PAB involves a spontaneous 1,6-elimination reaction (Jain et al., Pharm Res 2015, 32: 3526-3540):

[0276] In some embodiments, the cleavable linker comprises a Mal-spacer and a cleavable peptide portion. In some embodiments, the spacer comprises a PEG portion (e.g., 2 PEGs) and the cleavable peptide portion comprises an amino acid unit (e.g., a dipeptide VC and a tetrapeptide GGFG). In some embodiments, the cleavable linker comprises a covalently linked Mal-spacer-amino acid unit, wherein the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG. In some embodiments, the cleavable linker comprises a covalently linked Mal-spacer-amino acid unit-PAB, wherein the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG. In some embodiments, the spacer is (PEG) m , m is an integer of 1-5, preferably 2. In some embodiments, the cleavable linker comprises Mal-(PEG)2 and VC. In some embodiments, the cleavable linker comprises Mal-(PEG)2 and GGFG.

[0277] In some embodiments, the cleavable linker comprises the structure: Mal-spacer-cleavable peptide moiety. In some embodiments, the cleavable linker comprises the structure: Mal-(PEG)2-VC and Mal-(PEG)2-GGFG.

[0278] In some embodiments, the cleavable linker comprises a Mal-spacer, a cleavable peptide portion, and a second spacer. In some embodiments, the spacer comprises a PEG portion (e.g., 2 PEGs), the cleavable peptide portion comprises an amino acid unit (e.g., a dipeptide VC), and the second spacer is self-immolative (e.g., PAB). In some embodiments, the cleavable linker comprises Mal-(PEG)2, VC, and PAB.

[0279] In some embodiments, the cleavable linker comprises the structure: Mal-spacer-cleavable peptide moiety-second spacer. In some embodiments, the cleavable linker comprises the structure: Mal-(PEG)2-VC-PAB.

[0280] In some embodiments, the Mal-spacer of the cleavable linker is coupled to one or more amino acid residues of the antibody portion of the ADC of the present invention. For example, the Mal-spacer can be coupled to the antibody via a thiol group, including coupling to the sulfhydryl group of one or more cysteine ​​residues of the antibody. In some embodiments, the maleimide group of the Mal-spacer reacts with the sulfhydryl group of the cysteine ​​residue of the antibody, thereby conjugating the Mal-spacer to the antibody. In some embodiments, the maleimide group of the Mal-spacer can react with the sulfhydryl group of the cysteine ​​residue at a specific position in the constant region and / or variable region of the antibody. Further, the maleimide group of the Mal-spacer can be coupled to the free cysteine ​​residue released after reduction of the disulfide bonds and / or interchain disulfide bonds in the hinge region of the antibody. In some embodiments, the maleimide group of the Mal-spacer is coupled to the cysteine ​​residue in the hinge region of the antibody. The interchain disulfide bonds in the hinge region of the antibody can be subjected to a chemical reaction (e.g., reduction, pH adjustment, or hydrolysis) to generate a plurality of free cysteine ​​residues. Alternatively, the amino acid residues at one or more specific sites in the constant region of the antibody can be engineered to generate cysteine ​​residues through DNA recombination technology (e.g., by substitution or insertion of cysteine ​​residues).

[0281] In some embodiments, the second spacer of the cleavable linker is coupled to a small molecule compound, which is eribulin or a derivative thereof. In some embodiments, the second spacer couples the cleavable peptide portion of the cleavable linker to the C-35 amine of eribulin, and the second spacer is self-immolative (e.g., PAB), wherein the cleavable peptide portion comprises VC. The self-immolative spacer spontaneously undergoes self-immolative fragmentation after the cleavable peptide portion is enzymatically hydrolyzed, resulting in the release of eribulin from the ADC in its natural active form. In some embodiments, the self-immolative spacer is PAB, and the cleavable peptide portion VC is conjugated to the C-35 amine of eribulin to construct an ADC comprising the structure of Mal-(PEG)2-VC-PAB-Eribulin.

[0282] In some embodiments, the cleavable portion of the cleavable linker (e.g., a cleavable polypeptide) can be directly conjugated to the small molecule toxin compound portion of the ADC, wherein the small molecule toxin compound is eribulin or a derivative thereof. In some embodiments, the cleavable polypeptide comprises an amino acid unit comprising GGFG, which is conjugated to the C-35 amine of eribulin via the carboxyl group to construct an ADC comprising the structure Mal-(PEG)2-GGFG-eribulin. Direct conjugation of the GGFG tetrapeptide in the cleavable linker to eribulin also ensures that the cleavable linker of the ADC, upon cleavage within target cells, releases eribulin intact in its naturally active form, enabling it to exert targeted tumor cell killing and / or a bystander effect.

[0283] In some embodiments, the ADC structure of the present invention comprises the anti-HER2 complementary bispecific antibody of the present invention conjugated to eribulin or its derivative via a cleavable linker, wherein the cleavable linker comprises a Mal-spacer, a cleavable peptide portion, and / or a second spacer. In some embodiments, the ADC structure comprises the anti-HER2 complementary bispecific antibody of the present invention conjugated to the C-35 amine of eribulin via a cleavable linker, wherein the cleavable linker comprises Mal-(PEG)2-VC-PAB or Mal-(PEG)2-GGFG, and the free cysteine ​​residue released after reduction of the disulfide bonds and / or interchain disulfide bonds in the hinge region of the antibody is conjugated to the Mal of the cleavable linker.

[0284] In some embodiments, the ADC of the present invention comprises the following parts:

[0285] In some embodiments, the ADC of the present invention comprises an anti-HER2 complementary bispecific antibody of the present invention coupled to a drug payload, wherein the drug payload comprises Mal-(PEG)2-VC-PAB-Eribulin and Mal-(PEG)2-GGFG-Eribulin. In some embodiments, the ADC comprises an anti-HER2 complementary bispecific antibody of the present invention conjugated to Mal of the drug payload via its cysteine ​​residue, and further, the free cysteine ​​residue released after reduction of the disulfide bonds and / or interchain disulfide bonds in the hinge region of the complementary bispecific antibody is conjugated to Mal of the drug payload.

[0286] In some embodiments, the present invention provides an ADC having the formula:

[0287] Wherein, Ab represents the anti-HER2 complementary bispecific antibody of the present invention, and p is 2 to 8.

[0288] The p in the formula (I) is also referred to herein as the drug-to-antibody ratio (DAR) or the number of payload molecules, that is, the number of small molecule compounds coupled to the antibody, and the p is 2 to 8, for example, 4 to 8. The DAR value of the anti-HER2 complementary bispecific antibody contained in the ADC of the present invention, which is conjugated to Eribulin through a cleavable linker, has desirable characteristics, that is, the ADC not only has sufficient killing activity, but also has sufficient stability. In general, a higher DAR value (for example, p>8) may cause the ADC to aggregate and precipitate in an aqueous environment due to hydrophobic effects due to the strong hydrophobicity of the small molecule toxin compound, and may even cause safety risks in vivo; a lower DAR value (for example, p<2) may result in lower ADC activity against tumor cells and affect the efficacy. In some embodiments, the optimal DAR value is about 4 to 8. The average DAR value (i.e., the average number of payload molecules or average p) of the anti-HER2 complementary bispecific ADC of the present invention can be obtained by calculation using the results of conventional analytical methods known in the art (e.g., reverse phase LC-MS mass spectrometry and / or HIC-HPLC).

[0289] 4. Method for preparing the antibody of the present invention

[0290] 4.1 Polynucleotides, Vectors, and Host Cells

[0291] In one aspect, the present invention provides a nucleic acid encoding an anti-HER2 antibody or antigen-binding fragment thereof, or an anti-HER2 complementary bispecific antibody. The present invention also includes polynucleotide variants encoding the amino acid sequences described herein.

[0292] The amino acid sequence corresponding to the present invention, the probe or primer used for nucleic acid isolation, or the nucleotide sequence provided by the database for query can be obtained by back translation of the amino acid sequence. The polymerase chain reaction (PCR) program can be used to isolate and amplify the DNA sequence encoding the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 complementary bispecific antibody of the present invention. Oligonucleotides that define the desired ends of the DNA fragment combination are used as 5' and 3' primers. The oligonucleotides may further contain a recognition site for a restriction endonuclease to facilitate the insertion of the amplified DNA fragment combination into the expression vector. PCR technology is described in Saiki et al., Science 1988, 239: 487-491; Wu et al., ed., Recombinant DNA Methodology, 1989 Academic Press, pp. 189-196; Innis et al., ed., PCR Protocols: A Guide to Methods and Applications, 1990 Academic Press.

[0293] The nucleic acid molecules of the present invention include single-stranded and double-stranded forms of DNA and RNA, as well as corresponding complementary sequences, including isolated nucleic acid molecules, preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that allows identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, 2nd edition, Cold Spring Harbor Laboratory). Preferably, such sequences include open reading frames interrupted by internal non-translated sequences or introns that are not commonly found in eukaryotic genes and are provided and / or constructed. Sequences of non-translated DNA may be present 5' or 3' to the open reading frame, where such sequences do not interfere with manipulation or expression of the coding region.

[0294] The anti-HER2 antibodies or antigen-binding fragments thereof, or anti-HER2 complementary bispecific antibodies of the present invention are prepared by the following steps: site-specific mutagenesis of nucleotides in DNA encoding the anti-HER2 antibodies or antigen-binding fragments thereof, or anti-HER2 complementary bispecific antibodies using PCR mutagenesis or other techniques known to those of ordinary skill in the art to generate DNA encoding variants, and then expressing the recombinant DNA in cell culture as outlined herein. In addition, anti-HER2 antibodies or antigen-binding fragments thereof and anti-HER2 complementary bispecific antibodies can also be prepared by in vitro synthesis using established techniques.

[0295] As known to those skilled in the art, due to the degeneracy of the genetic code, the anti-HER2 antibodies or antigen-binding fragments thereof or anti-HER2 complementary bispecific antibodies of the present invention are encoded by a very large number of nucleic acids, each of which is within the scope of the present invention and can be prepared using standard techniques. Therefore, once a specific amino acid sequence has been identified, those skilled in the art can simply modify one or more of the above codons in their respective coding sequences without changing the amino acid sequence of the anti-HER2 antibodies or antigen-binding fragments thereof or anti-HER2 complementary bispecific antibodies of the present invention to prepare a number of different nucleic acids.

[0296] On the other hand, the present invention also provides an expression vector for the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody described herein.

[0297] The nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof, or anti-HER2 complementary bispecific antibody of the present invention can be constructed into a suitable vector and introduced into a host cell for expression of the target protein. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the target protein in the vector is operably linked to the promoter.

[0298] As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0299] Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist its entry into cells, including but not limited to viral particles, liposomes, or protein shells.

[0300] In another aspect, the present invention further provides a host cell comprising a nucleic acid or expression vector encoding the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody of the present invention.

[0301] The cell can be a eukaryotic cell, for example, a mammalian host cell, including but not limited to, SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J Gen Virol 1977, 36:59-74), baby hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), mouse testicular Sertoli cells (TM4, Mather, Biol Reprod 1980, 23:243-251), monkey kidney cells (CV1, ATCC, CCL-70), African green monkey kidney cells (VERO-76, ATCC, CRL-1587), human cervical cancer cells (HELA, ATCC, CCL-2), canine kidney cells (MDCK, ATCC, CCL-34), Buffalo rat liver cells (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human hepatoma cell line (HepG2, ATCC, HB-8065), mouse mammary tumor (MMT060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC5 cells, or FS4 cells. 4.2 Screening of anti-HER2 antibodies of the present invention

[0302] The anti-HER2 antibodies provided by the present invention may be murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for producing monoclonal antibodies are known in the art, and any known method (e.g., hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.) may be used in the present invention to prepare monoclonal antibodies that specifically bind to HER2.

[0303] In some embodiments, a polypeptide of any fragment in the HER2 amino acid sequence (preferably the extracellular region of HER2) can be selected as the target antigen to immunize mice, and the antibody variable region genes can be amplified from mature B cells of the mice. The mouse antibody variable regions are displayed on the surface of phages by phage display technology to construct a phage display library. Panning is performed using specific target molecules (e.g., the target antigen used to immunize mice), and the interaction between the antibody variable regions displayed on the phage surface and the target antigen is detected. The antibody variable region library is screened and amplified by an in vitro selection method, which is similar to natural selection.

[0304] In some embodiments, the present invention screens antibodies that recognize the target antigen from the antibody library by constructing a phage display library and performing panning for the target antigen. The antibodies of the present invention can be produced using a variety of phage display methods known in the art. For example, US5223409, US5622699, and US6068829 disclose methods for preparing phage libraries. Phage display libraries can also be constructed according to the method described in "Antibody Phage Display: Methods and Protocols (O'Brien and Aitken ed.)". In some embodiments, the nucleic acid encoding the antibody variable region can be inserted into the phage coat protein gene so that the phage displays the antibody variable region on its surface. At the same time, the nucleic acid encoding the antibody variable region is contained inside the phage, thereby realizing the connection between the antibody variable region phenotype and genotype.

[0305] Regarding the screening of antibodies (or single-chain antibodies), in phage display, a large library of VH and / or VL regions of antibodies (or single-chain antibodies) can be expressed on the surface of filamentous phage particles, thereby pairing them to form binding domains. Phage can be selected from the library based on recognition and binding to the target antigen and the binding domain displayed.

[0306] In some embodiments, the panning can be achieved by phage infection of host bacteria and propagation and amplification in the host. Phages secreted by the host bacteria and displaying single-chain antibody fragments on their surfaces are collected and, as needed, subjected to multiple rounds of panning until phages that selectively or specifically bind to the target antigen are obtained. Finally, the amino acid sequence of the antibody variable region is obtained by sequencing the antibody gene in the phage genome (Arap et al., Science 1998, 279: 377-380; Smith et al., Science 1985, 228: 1315-1317).

[0307] In a specific embodiment, the present invention uses a HER2 extracellular domain recombinant protein as the target antigen to immunize mice, obtains antibody heavy chain variable region family and light chain variable region family gene fragments from the spleen tissue of the immunized mice, and produces scFv fragments based on these fragments. These fragments are then linked to the phage surface structural protein gene III. Using a co-expression method, these fragments participate in phage assembly and are displayed on the phage surface, thereby constructing a phage display library. Using the phage display library, for a specific target antigen (e.g., the antigen used to immunize mice), through multiple rounds of adsorption-elution-amplification repetitive processes (panning), phages that can specifically bind to the target antigen can be enriched. Gene sequencing technology is then used to obtain the corresponding DNA sequence information, and the amino acid sequence of the antibody variable region can be inferred.

[0308] 5. Preparation method

[0309] 5.1 Methods for preparing anti-HER2 antibodies or antigen-binding fragments thereof, or anti-HER2 complementary bispecific antibodies

[0310] The present invention provides a method for preparing the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody of the present invention using the host cell.

[0311] The method comprises transfecting a nucleic acid or expression vector encoding an anti-HER2 antibody or antigen-binding fragment thereof, or an anti-HER2 complementary bispecific antibody of the present invention into a host cell, and culturing the host cell in a culture medium for a period of time to express the anti-HER2 antibody or antigen-binding fragment thereof, or an anti-HER2 complementary bispecific antibody of the present invention. Without limitation, commercially available culture medium can be used as the cell culture medium.

[0312] Preferably, the expressed anti-HER2 antibody or antigen-binding fragment thereof, or anti-HER2 complementary bispecific antibody can be secreted into the culture medium in which the host cells are grown. The antibody is recovered from the culture medium using conventional protein purification methods, such as removing impurities by centrifugation or ultrafiltration, or purifying the resulting product by affinity chromatography; other purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography, can also be used.

[0313] 5.2 Preparation of Anti-HER2 Complementary Bispecific ADCs

[0314] The ADC of the present invention can be prepared by any method known in the art, including but not limited to: (1) reacting a nucleophilic group or an electrophilic group of an antibody with a cleavable linker to form an antibody-cleavable linker intermediate (Ab-L) via a covalent bond, and then reacting with a small molecule toxin compound (D), wherein the intermediate Ab-L may or may not undergo a purification step before reacting with the small molecule toxin compound; (2) reacting a nucleophilic group or an electrophilic group of a small molecule toxin compound with a cleavable linker to form a small molecule toxin compound-cleavable linker intermediate (DL) via a covalent bond, and then reacting with a nucleophilic group or an electrophilic group of an antibody, wherein the intermediate DL may or may not undergo a purification step before reacting with the antibody; or (3) the antibody, the cleavable linker, and the small molecule toxin compound can be mixed and reacted so that covalent bonds are formed simultaneously between the antibody and the cleavable linker and between the cleavable linker and the small molecule toxin compound to prepare the ADC of the present invention. Several specific examples of methods for preparing ADCs are known in the art, such as those described in US Pat. No. 8,624,003 (one-pot method), US Pat. No. 8,163,888 (one-step method), and US Pat. No. 5,208,020 (two-step method).

[0315] In some embodiments, the antibody is placed under reducing conditions before the conjugation reaction to produce one or more free cysteine ​​residues. In some embodiments, a reducing agent (e.g., dithiothreitol [DDT], 2-mercaptoethanol or tris(2-carboxyethyl)phosphine [TCEP]) is used to act on the antibody to reduce the interchain disulfide bonds in the hinge region of the antibody to produce partially or completely reduced antibodies with free sulfhydryl groups. The reducing agent preferentially reduces the interchain disulfide bonds in the hinge region of the antibody, while the intrachain disulfide bonds of the antibody remain intact. In some embodiments, in a buffer containing a chelating agent, the reducing agent TCEP is used to react with the antibody to obtain a partially or completely reduced antibody with free sulfhydryl groups. The chelating agent includes but is not limited to ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), and the buffer includes but is not limited to histidine-hydrochloric acid, sodium phosphate, sodium borate, and sodium acetate solution.

[0316] In some embodiments, the partially or completely reduced antibody with free sulfhydryl groups obtained above can be reacted with a reactive functional group (e.g., maleimide group) of a cleavable linker (L) or a small molecule toxin compound-cleavable linker intermediate (DL) to form a covalent bond (e.g., a thioether bond) to obtain an antibody-cleavable linker intermediate (Ab-L) or ADC. Herein, the small molecule toxin compound-cleavable linker intermediate (DL) is used interchangeably with the drug payload.

[0317] The purification method of the ADC prepared by the above method can be any biochemical method known in the art for purifying proteins or any combination thereof, including but not limited to affinity chromatography, ion exchange chromatography, mixed mode chromatography (e.g., ceramic hydroxyapatite chromatography), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

[0318] 6. Pharmaceutical Composition

[0319] The present invention provides a pharmaceutical composition comprising the anti-HER2 antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, or the anti-HER2 complementary bispecific ADC according to the present invention, and a pharmaceutically acceptable carrier.

[0320] The pharmaceutical composition can include any type of pharmaceutically acceptable carrier. Operable carriers include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. For example, the following works teach the selection and use of suitable carriers: Gennaro, Remington: The Science and Practice of Pharmacy, 2003 20th edition (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.

[0321] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration. " Parenteral administration " as used in the present invention refers to non-enteral and local mode of administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibody of the present invention can also be administered by non-parenteral route (for example, local, epidermal or mucosal administration route), for example, intranasal, oral, vaginal, rectal, sublingual or topical administration. In view of different routes of administration, the active ingredient can be coated in the material to protect it from the effect of acid / alkali and other natural conditions that may inactivate it.

[0322] The pharmaceutical compositions can be sterile aqueous solutions or dispersions. They can also form microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentration.

[0323] The amount of active ingredient that can be combined with a carrier material to form a single dosage form is determined based on the subject and the particular mode of administration, and is generally an amount of the composition that will produce a therapeutic effect. Typically, the composition formed with a pharmaceutically acceptable carrier contains from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%.

[0324] The dosage regimen is adjusted to provide the best expected response (e.g., a therapeutic response). For example, a single dose may be administered, a divided dose may be administered, or the dose may be reduced or increased proportionally depending on the treatment situation. It is particularly advantageous to formulate a composition for parenteral administration in a dosage unit form, which facilitates administration and uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit that is suitable as a unit dose for treating a subject; each unit dose contains a predetermined amount of active ingredient, and the predetermined amount of the active ingredient is obtained by calculating the active ingredient together with the desired pharmaceutical carrier to produce the expected therapeutic effect. In addition, the anti-HER2 antibody, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC of the present invention can also be administered as a sustained-release formulation, which can reduce the frequency of administration.

[0325] The dosage of the anti-HER2 antibody or antigen-binding fragment thereof, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC of the present invention, or the composition comprising the anti-HER2 antibody or antigen-binding fragment thereof, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC can be in the range of about 0.0001 mg / kg to 100 mg / kg body weight, typically 0.001 mg / kg to 50 mg / kg body weight.

[0326] The "therapeutically effective dose" of the anti-HER2 antibody or antigen-binding fragment thereof, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC of the present invention preferably reduces the severity of disease symptoms, increases the frequency and duration of disease symptom-free periods, or prevents damage to the body or disability caused by the disease. For example, a "therapeutically effective dose" in a tumor-bearing subject preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, relative to an untreated subject. A therapeutically effective amount of a therapeutic antibody or ADC can reduce the size of a tumor or improve the symptoms of a subject, who is typically a human or other mammal. The "therapeutically effective dose" can also be determined differently based on various factors, including but not limited to formulation method, administration method, age, body shape, weight, patient's sex or pathological condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity.

[0327] Pharmaceutical compositions can be selected from controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, 1978 (Marcel Dekker).

[0328] The therapeutic drug composition can be delivered by a medical device selected from the group consisting of: (1) needle-free subcutaneous injection devices (e.g., US5399163, US5383851, US5312335, US5064413, US4941880, US4790824, and US4596556); (2) microinfusion pumps (US4487603); (3) transdermal devices (US4486194); (4) infusion sets (US4447233 and US4447224); and (5) osmotic devices (US4439196 and US4475196); the disclosures of which are incorporated herein by reference.

[0329] In certain embodiments, the anti-HER2 antibodies or antigen-binding fragments thereof, anti-HER2 complementary bispecific antibodies, or anti-HER2 complementary bispecific ADCs of the present invention can be formulated to ensure biodistribution in vivo. For example, to ensure that the therapeutic antibodies or ADCs of the present invention cross the blood-brain barrier, they can be formulated into liposomes, which can further comprise a targeting moiety to enhance selective delivery to specific cells or organs. See, e.g., U.S. Pat. No. 4,522,811, U.S. Pat. No. 5,374,548, U.S. Pat. No. 5,416,016, and U.S. Pat. No. 5,399,331; Ranade, J Clin Pharmacol 1989, 29:685-694; Umezawa et al., Biochem Biophys Res Commun 1988, 153:1038-1044; Bloeman et al., FEBS Lett 1995, 357:140-144; Owais et al., Antimicrob Agents Chemother 1995, 39:180-184; Briscoe et al., Am J Physiol 1995, 268:L374-380; Schreier et al., J Biol Chem 1994, 269:9090-9098; Keinanen and Laukkanen, FEBS Lett 1994, 346:123-126; and Killion and Fidler, Immunomethods 1994, 4:273-279.

[0330] 7. Medicine Box

[0331] The present invention provides a drug kit comprising an effective amount of an anti-HER2 antibody or antigen-binding fragment thereof, an anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific ADC, or a pharmaceutical composition of the present invention, and optionally at least one additional tumor therapeutic agent (i.e., the drug kit may contain or not contain at least one additional tumor therapeutic agent). Preferably, the tumor therapeutic agent may include but is not limited to another antagonist of ErbB2 / HER2; an EGFR antagonist, a HER3 antagonist; a MET antagonist, a small molecule inhibitor of MET (e.g., Capmatinib); an IGF1R antagonist (e.g., an anti-IGF1R antibody); a B-Raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720); a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody); a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody or a small molecule agonist). enzyme inhibitors [e.g., imatinib mesylate or sunitinib malate]); PDGF ligand inhibitors (e.g., anti-PDGF-A antibody, anti-PDGF-B antibody, anti-PDGF-C antibody or anti-PDGF-D antibody, aptamer, siRNA, etc.); VEGF antagonists (e.g., VEGF-Trap, such as aflibercept); anti-VEGF antibodies (e.g., bevacizumab); VEGF receptor kinase inhibitors (e.g., sunitinib, sorafenib or pazopanib); DLL4 antagonists (e.g., REGN421); Ang2 antagonists (e.g., WO20 11014469 disclosed anti-Ang2 antibodies, such as H1H685P); FOLH1 antagonists (e.g., anti-FOLH1 antibodies); STEAP1 or STEAP2 antagonists; TMPRSS2 antagonists; MSLN antagonists; MUC16 antagonists; CLEC12A antagonists; PD-1 or PD-L1 blockers (e.g., pembrolizumab or nivolumab); hormone receptor modulators (e.g., estrogen receptor modulators [such as tamoxifen]; androgen receptor modulators); aromatase inhibitors (e.g., letrozole); , anastrozole, exemestane); kinase inhibitors (e.g., tyrosine kinase inhibitors [such as lapatinib]); cytokine agonists; cytokine inhibitors (including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18 or their respective receptors); chemotherapeutic agents (including but not limited to microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents), etc.

[0332] In some embodiments, the other ErbB2 / HER2 antagonist includes an anti-HER2 monoclonal antibody (e.g., Trastuzumab, Pertuzumab) and / or ADC (e.g., T-DM1, DS-8201), or an anti-ErbB2 / HER2 small molecule inhibitor (e.g., tyrosine kinase inhibitors such as lapatinib, pyrotinib, and neratinib); the EGFR antagonist includes an anti-EGFR antibody (e.g., Cetuximab, Panitumumab), an anti-EGFR small molecule inhibitor (e.g., Gefitinib, Erlotinib), an anti-EGFRvIII antagonist (e.g., anti-EGFRvIII antibody); the HER3 antagonist includes but is not limited to an anti-HER3 antibody (e.g., Patritumab); the MET antagonist includes but is not limited to an anti-MET antibody (e.g., Onatuzumab, Ematuzumab, and H4H14639D).

[0333] 8. Methods and uses for treating HER2-expressing cancers

[0334] In one aspect, the present invention relates to a method for treating a HER2-expressing cancer, comprising administering to a subject in need thereof an effective amount of an anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific ADC, or an inventive pharmaceutical composition or kit. Alternatively, the present invention relates to the use of the above-described antibody, ADC, pharmaceutical composition, or kit in the preparation of a medicament for treating a HER2-expressing cancer. Alternatively, the present application relates to the above-described antibody, ADC, pharmaceutical composition, or kit for treating a HER2-expressing cancer.

[0335] The cancer includes, but is not limited to, breast cancer, ovarian cancer, cervical cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, melanoma, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, and endometrial cancer. The cancer also includes cancers at various stages, such as early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The cancer can be HER2 overexpressed or underexpressed. The subject can be a human, non-human primate, or other mammal such as dog, mouse, or rat.

[0336] As is known in the art, cancers expressing HER2 can be characterized by the amount of HER2 expressed on the surface of cancer cells (i.e., by "HER2 status"). HER2 expression can be assessed by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and the like. In some embodiments, the cancer includes a cancer that overexpresses HER2 (e.g., IHC 3 + , or IHC 2 + / FISH+ ), and / or HER2-low expressing cancers (IHC 2 + / FISH - , or IHC 1 + ).

[0337] In some embodiments, the anti-HER2 complementary bispecific ADC or its pharmaceutical composition has a killing effect on both HER2-overexpressing tumors / cancers and / or HER2-underexpressing tumors / cancers.

[0338] In some embodiments, the anti-HER2 complementary bispecific antibody or pharmaceutical composition thereof has inhibitory activity on the growth or proliferation of tumor cells overexpressing HER2.

[0339] In some embodiments, administering an effective amount of an anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, pharmaceutical composition, or kit of the invention to a subject in need thereof can reduce the subject's tumor volume, inhibit tumor growth, prolong the subject's disease-free survival or progression-free survival, increase the subject's overall survival, reduce tumor metastasis, or improve the subject's quality of life.

[0340] In another aspect, the present invention also relates to a method for treating cancer that has become resistant to or relapsed from existing HER2-targeted therapeutics, comprising administering to a patient in need thereof an effective amount of an anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, pharmaceutical composition, or kit of the present invention. Alternatively, the present invention relates to the use of the antibody, ADC, pharmaceutical composition, or kit in the preparation of a medicament for treating cancer that has become resistant to or relapsed from HER2-targeted therapeutics.

[0341] In some embodiments, the patient does not respond or responds poorly to one or more existing HER2 targeted therapeutics, including trastuzumab, pertuzumab, T-DM1, and DS-8201. The non-response is manifested as tumor growth, increased tumor volume, increased tumor metastasis formation, or an increase in the number of metastases in the patient. The non-response can also be a shortened development of tumor metastasis or disease progression time. The poor response refers to the growth or metastasis of the patient's tumor within a short period of time during or after the standard treatment of a HER2 targeted therapeutic.

[0342] In another aspect, the present invention further relates to a method for treating patients who are not suitable for existing HER2 targeted therapies or who are refractory to treatment, or who develop drug resistance or relapse after receiving existing HER2 targeted therapies, the method comprising administering to a patient in need thereof an effective amount of an anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific ADC, a pharmaceutical composition, or a kit of medicine of the present invention. Alternatively, the present invention relates to the use of the antibody, ADC, pharmaceutical composition, or kit of medicine for preparing a medicament for treating patients who are not suitable for existing HER2 targeted therapies or who are refractory to treatment, or who develop drug resistance or relapse after receiving existing HER2 targeted therapies. The HER2 targeted therapies include treatment with trastuzumab, pertuzumab, T-DM1, or DS-8201.

[0343] In some embodiments, when a patient has experienced disease progression in response to existing HER2-targeted therapies, the anti-HER2 complementary bispecific antibodies, anti-HER2 complementary bispecific ADCs, pharmaceutical compositions, or kits of the present invention can be used to treat progressive cancer.

[0344] In another aspect, the anti-HER2 complementary bispecific antibodies, anti-HER2 complementary bispecific ADCs, pharmaceutical compositions, or kits of the present invention can be used alone or in combination with other types of cancer therapies known in the art, such as surgery, chemotherapy, radiotherapy, gene therapy, immunotherapy, photodynamic therapy, radiofrequency ablation, and the like.

[0345] 9. Detection purpose

[0346] The present invention also relates to the use of the anti-HER2 antibody or its antigen-binding fragment or the anti-HER2 complementary bispecific antibody for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, and for screening cancer patients who are responsive to the anti-HER2 complementary bispecific ADC treatment of the present invention. Alternatively, the present application relates to a method for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, and a method for screening cancer patients who are responsive to the above-mentioned ADC treatment, comprising incubating the anti-HER2 antibody or its antigen-binding fragment or the anti-HER2 complementary bispecific antibody with the sample or a biological sample separated from the patient, and detecting whether the antibody is bound to the sample or biological sample. Alternatively, the present application relates to the anti-HER2 antibody or its antigen-binding fragment or the anti-HER2 complementary bispecific antibody for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, or screening cancer patients who are responsive to the above-mentioned ADC treatment.

[0347] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 complementary bispecific antibody can be used to diagnose a condition or disease of abnormal HER2 expression (e.g., overexpression, underexpression, or lack of expression) to facilitate determining a treatment regimen. For example, the antibody can be conjugated with a detectable marker or reporter molecule, and the labeled antibody can be contacted with a sample obtained from a patient to diagnose HER2 expression. The detectable marker or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S, or 125 I; fluorescent materials, for example, umbelliferone, luciferin, rhodamine, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; chemiluminescent materials, for example, luminol; bioluminescent materials, for example, luciferase, luciferin or aequorin; or enzymes, for example, alkaline phosphatase, β-galactosidase, acetylcholinesterase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure HER2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoPET (e.g., 89 Zr, 64 Cu, etc.) and fluorescence activated cell cytometry (FACS).

[0348] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all drawings and all references, patents and patent applications cited in the present invention are expressly incorporated herein by reference. Unless otherwise noted, the materials, reagents and devices involved in the following examples are commercially available. Example

[0349] Example 1. Construction of anti-HER2 immune library and screening of antibodies

[0350] 1.1 Construction of anti-HER2 immune library

[0351] Two BALB / c mice were immunized with human HER2 extracellular domain recombinant protein (sequence derived from position 23-652 of NP_004439.2). After three immunizations, blood was collected from the mice and the immune titer was tested using ELISA. The results showed that the immune titer was above 1:1,000,000 (data not shown). The mice were boosted with immunization, and the spleens were removed under sterile conditions three days later, and a single cell suspension of the mouse spleen was prepared. The collected mouse spleen cells were lysed using the Animal Total RNA Isolation Kit (FuGene) and RNA was extracted; the extracted RNA was used as a template and reverse transcription was performed using a PrimeScript Reverse Transcription Kit (PrimeScript TM cDNA was obtained by reverse transcription using a PCR amplification kit (RT-PCR) using specific primers. The cDNA was then used as a template to amplify the gene fragments of the heavy and light chain variable regions of the mouse antibody family. The light and heavy chain variable region gene fragments were then spliced ​​and amplified by overlapping PCR (SOE-PCR) to obtain single-chain antibody variable region gene fragments (scFv). The scFv sequence was ligated into a phagemid display vector by enzyme digestion. The ligation product was then electroporated into TG1 competent cells, and the bacterial solution was infected with helper phage M13KO7 to obtain two phage display libraries.

[0352] 1.2 Screening and sequence analysis of anti-HER2 antibodies

[0353] The two phage display libraries described in Item 1.1 of this Example were panned using the HER2 extracellular domain recombinant protein as the target antigen. After 3 to 5 rounds of "adsorption-elution-amplification" panning, monoclonal ELISA screening and PCR enzyme digestion assay verification, and sequencing analysis of the candidate clones, 15 sequence-specific anti-HER2 murine monoclonal antibodies were finally obtained.

[0354] 1.3 Preparation of anti-HER2 chimeric antibodies

[0355] The light and heavy chain genes of the 15 mouse antibodies obtained by the above screening were homologously recombined with the linearized expression vector pcDNA3.1 (the heavy chain variable region [VH] was connected to the pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region [VL] was connected to the pcDNA3.1 vector containing the human CK constant region). The chimeric antibody expression vector with the correct sequence was obtained by colony PCR detection and DNA sequencing analysis. The chimeric antibody expression vector was extracted and purified using conventional methods. Then, the 15 groups of chimeric antibody expression vectors containing light and heavy chain gene fragments were transiently transformed into ExpiCHO-S cells (Thermo). The chimeric antibodies were expressed by serum-free culture and purified by Protein A affinity chromatography using the AKTA Pure system to obtain anti-HER2 chimeric antibody protein.

[0356] 1.4 Detection of binding activity of anti-HER2 chimeric antibodies to ErbB / HER family members

[0357] The binding activity of anti-HER2 chimeric antibodies to human ErbB / HER family members was detected by ELISA. The specific method is as follows: dilute the HER2 extracellular domain recombinant protein and the extracellular domain recombinant proteins of other members of the human ErbB / HER family (EGFR, HER3 and HER4) with PBS, and then add 100 μL / well to a 96-well ELISA plate and incubate at 4°C overnight. After washing the plate with PBS, add 200 μL of blocking solution (PBST containing 1% BSA [PBS + 0.1% Tween-20]) to each well and incubate at room temperature for 1 hour. After washing the plate with PBST, add 100 μL of the test antibodies (including anti-HER2 chimeric antibodies and control antibodies Trastuzumab, Cetuximab or Patritumab) diluted in the blocking solution to each well and incubate at room temperature for 2 hours. After washing with PBST, 100 μL of anti-human IgG (H+L)-HRP antibody (Jackson Immuno) diluted 1:10,000 in blocking buffer was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of TMB colorimetric solution was added to each well and incubated at room temperature in the dark for 3-10 minutes. Then, 50 μL of stop solution (2 M HCl) was added to each well, and OD450 values ​​were read using a multi-function microplate reader (Varioskan, Thermo). Raw data were analyzed using GraphPad Prism 9 software. As shown in Figure 1, mAb2117, mAb2126, mAb2128, mAb2133, mAb2138, mAb2164, and mAb2170 showed strong binding activity to HER2 and showed no cross-binding activity with other members of the human ErbB / HER family.

[0358] 1.5 Detection of binding activity of anti-HER2 chimeric antibodies to HER2-expressing tumor cells

[0359] The binding activity of anti-HER2 chimeric antibodies to NCI-N87 cells was detected by flow cytometry. The specific method was as follows: NCI-N87 cells were collected, washed once with pre-cooled FACS buffer (PBS + 1% BSA), and then resuspended to 6-10×10 6 Cells were plated at 50 μL / well in a 96-well U-shaped plate. 50 μL / well of serially diluted chimeric antibodies to be tested or positive control antibody trastuzumab (FACS buffer was used as a negative blank control) were then added and incubated on ice for 60 minutes. After washing the cells twice with pre-chilled FACS buffer, 100 μL / well of anti-human IgG (H+L)-AF488 antibody (Jackson Immuno) diluted 1:1,000 in FACS buffer was added and incubated on ice for 40 minutes in the dark. After washing the cells twice with pre-chilled FACS buffer, 100 μL / well of pre-chilled FACS buffer was added to resuspend the cells and analyzed by flow cytometry (NovoCyte 3005, Agilent). As shown in Figure 2, mAb2117, mAb2126, mAb2164, and mAb2170 antibodies showed strong binding activity to NCI-N87 cells.

[0360] 1.6 Antigen Binding Epitope Analysis of Anti-HER2 Chimeric Antibodies

[0361] According to the structure of the human and mouse HER2 extracellular subdomains, the gene sequences of different subdomains of human and mouse HER2 were recombined and spliced, and homologous recombination was connected to the pcDNA3.1 vector to construct a human-mouse chimeric HER2 extracellular region recombinant protein expression plasmid (Figure 3A), and transient expression was obtained to obtain the human-mouse chimeric HER2 extracellular region recombinant protein supernatant (the C-terminus of each recombinant protein was carrying a 6×His tag). The human-mouse chimeric recombinant protein was used as an antigen to detect the binding epitope of the chimeric antibody in the HER2 extracellular region by ELISA. The specific method is as follows: After the chimeric antibody to be tested was diluted with PBS, 100 μL / well was added to a 96-well ELISA plate and incubated at 4°C overnight. After washing the plate with PBS, 200 μL of blocking solution (PBST containing 1% BSA) was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of human HER2, mouse HER2, or human-mouse chimeric HER2 recombinant protein diluted 1:200 in blocking buffer was added to each well. These proteins include HER2(D1), HER2(D1-2), HER2(D1-3), HER2(D2), HER2(D3), and HER2(D4). The suffixes in parentheses indicate that the human sequence in one or more extracellular subdomains of the HER2 chimeric antigen has been replaced by the corresponding mouse sequence. For example, D1 indicates that extracellular subdomain 1 has been replaced by the mouse sequence. After incubation at room temperature for 2 hours, the plates were washed with PBST and 100 μL of anti-6×His-HRP antibody (Proteintech) diluted 1:10,000 in blocking buffer was added to each well. The cells were incubated at room temperature for 1 hour. After washing with PBST, 100 μL of TMB colorimetric solution was added to each well. After incubation at room temperature in the dark for 3-10 minutes, 50 μL of stop solution (2 M HCl) was added to each well. OD450 values ​​were then read using a multifunctional microplate reader (Varioskan, Thermo). Raw data were analyzed using GraphPad Prism 9 software. The test results are shown in Figure 3B. The recognition epitope of mAb2117 is located at D3 of the HER2 extracellular region, the recognition epitope of mAb2126, mAb2164, and mAb2170 is located at D1 of the HER2 extracellular region, and the recognition epitope of mAb2128 is located at D4 of the HER2 extracellular region.

[0362] 1.7 Antigen Binding Competition Analysis of Anti-HER2 Chimeric Antibodies

[0363] To further verify whether the epitopes bound by each chimeric antibody overlapped or overlapped with those of HER2, the antigen binding competition activity between the chimeric antibodies was tested by competitive ELISA. The specific method was as follows: 100 μL / well of the HER2 extracellular domain recombinant protein was diluted in PBS and added to a 96-well ELISA plate. The plate was incubated overnight at 4°C. After washing with PBS, 200 μL of blocking buffer (PBST containing 1% BSA) was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 50 μL / well of each chimeric antibody to be tested or the control antibody trastuzumab (starting at 50 μg / mL and followed by a 3-fold dilution series for a total of 7 concentrations) diluted in blocking buffer were added. After incubation at room temperature for 1 hour, 50 μL / well of each biotin-labeled chimeric antibody (final concentration of 0.5 μg / mL) was added to each well and incubated at room temperature for another 1 hour. After washing with PBST, 100 μL of HRP-Streptavidin (Jackson Immuno) diluted 1:10,000 in blocking buffer was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of TMB colorimetric solution was added to each well and incubated at room temperature in the dark for 3-10 minutes. After that, 50 μL of stop solution (2 M HCl) was added to each well, and the OD450 value was read using a multi-function microplate reader (Varioskan, Thermo). The test results showed that mAb2164 and mAb2170 completely competed for binding to the HER2 extracellular domain recombinant protein (Figure 4A), mAb2164 partially competed with mAb2117 and mAb2126 for binding to the HER2 extracellular domain recombinant protein, and mAb2164 did not compete with trastuzumab for binding to the HER2 extracellular domain recombinant protein (Figure 4A). mAb2164 did not compete with mAb2128 for binding to the HER2 extracellular domain recombinant protein, and mAb2117 did not compete with mAb2126 and mAb2128 for binding to the HER2 extracellular domain recombinant protein (Figure 4B). 1.8 Antigen Binding Kinetic Analysis of Anti-HER2 Chimeric Antibodies

[0364] The binding kinetics of each chimeric antibody to the HER2 extracellular domain recombinant protein were measured using a molecular interaction analyzer (ForteBio, model R8). The specific method was as follows: the chimeric antibodies mAb2117, mAb2126, mAb2128, mAb2164, and mAb2170 to be tested were diluted to 5 μg / mL in PBS and added to a 96-well black-walled plate at 200 μL per well. The HER2 extracellular domain recombinant protein was then diluted to 200 nM in PBS and serially diluted 1:2, before being added to a 96-well black-walled plate at 200 μL per well. The 96-well black-walled plate containing the test sample and the Protein A detection probe were placed in the analyzer. The program was set to allow the Protein A probe to capture the antibody, followed by binding and dissociation assays with the antigen. After each binding and dissociation cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe before the next cycle was repeated. After the test is completed, the analysis software is used to fit the binding constant and dissociation constant between the corresponding antibody and antigen, and the affinity constant (K D ) values. The results are shown in Table 4. The binding affinity constants (K D ) values ​​are all about 10 -9 M.

[0365] Table 4. Binding kinetic analysis results of chimeric antibodies and HER2 extracellular domain recombinant protein Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0366] 1.9 Effect of anti-HER2 chimeric antibodies on HER2 Y1248 phosphorylation

[0367] Published research results have shown that trastuzumab can induce phosphorylation of the Y1248 site of the HER2 intracellular domain in cardiomyocytes, thereby triggering a series of signal transduction cascades, ultimately leading to the disruption of cardiomyocyte homeostasis (Mohan et al., Molecular Cancer Therapeutics 2016, 15:1321-1331). This mechanism may contribute to the cardiotoxicity caused by trastuzumab. Therefore, this example evaluated whether anti-HER2 antibodies induce HER2-Y1248 phosphorylation in SKBR-3 cells to assess whether they exhibit cardiotoxicity similar to trastuzumab. The specific method is as follows: SKBR-3 cells were harvested and resuspended in RPMI-1640 serum-free medium (containing 0.1% BSA), then seeded into 6-well plates and cultured overnight at 37°C. The test antibody or control antibody (trastuzumab, pertuzumab) was added to each well at a final concentration of 4 μg / mL and incubated at 37°C for 30 minutes. Cells from each well were collected into a 1.5-mL EP tube and washed twice with ice-cold PBS. Total protein extraction was performed using 150 μL of RIPA cell lysis buffer (Porteintech). The supernatant was collected by high-speed centrifugation at 4°C for 10 minutes. Protein concentration in the supernatant was determined by BCA assay, and reducing buffer was added to prepare samples for protein electrophoresis. SDS-PAGE gel electrophoresis was then performed. Following electrophoresis, protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane at 80 V for 90 minutes. The membrane was then blocked with 5% skim milk at room temperature. Rabbit anti-human HER2 (Y1248) antibody (CST) or rabbit anti-human HER2 antibody (Abcam) was added at a 1:1000 dilution and incubated overnight at 4°C. The PVDF membrane was washed three times with PBST, and goat anti-rabbit IgG-HRP antibody (Jacskon Immuno) was added at a 1:5000 dilution in 5% skim milk and incubated for 1 hour at room temperature. The PVDF membrane was washed three times with PBST, followed by addition of developer and detection using an imager (Bio-Rad). As shown in Figure 5, compared to the positive control, trastuzumab, chimeric antibodies mAb2117 and mAb2126 did not induce phosphorylation of HER2-Y1248, suggesting that they may not exhibit the same cardiotoxicity as trastuzumab.

[0368] 1.10 Effect of anti-HER2 chimeric antibodies on NRG-1-induced activation of HER2 downstream signaling pathways

[0369] NRG-1 can induce heterodimerization of HER2 and activate downstream signal transduction pathways, among which AKT phosphorylation is a landmark event in its downstream signal transduction cascade. This example evaluates the effect of antibodies on the HER2 receptor signal transduction pathway by detecting their inhibitory effect on NRG-1-induced AKT phosphorylation. The specific method is as follows: T47D cells are seeded into 6-well plates. After the cells grow to about 80% confluence, the culture medium is discarded and serum-free RPMI-1640 is added for starvation culture overnight. The test antibody or control antibody (Trastuzumab, Pertuzumab) is added to each well at a final concentration of 100nM. After incubation at 37°C for 20 minutes, NRG-1 (NOVUS) is added to each well at a final concentration of 100ng / mL and incubated for another 10 minutes. After washing the cells twice with pre-chilled PBS, whole protein extraction and Western blot analysis were performed according to the method described in Section 1.9 of this Example. The primary antibodies used were rabbit anti-p-AKT (Ser465) antibody (CST) or rabbit anti-pan-AKT antibody (CST), and the secondary antibody was goat anti-rabbit IgG-HRP antibody (Jacskon Immuno). The results are shown in Figure 6. Compared with the positive control antibody pertuzumab, the chimeric antibodies mAb2117 and mAb2126 had no inhibitory effect on NRG-1-induced AKT phosphorylation, indicating that they do not affect NRG-1-induced HER2 heterodimerization and activation of its downstream signaling pathways.

[0370] Example 2 Humanization and Optimization of Anti-HER2 Chimeric Antibody mAb2117

[0371] 2.1 Humanization of the anti-HER2 chimeric antibody mAb2117

[0372] After analysis by MOE software, the heavy chain framework sequence of the chimeric antibody mAb2117 was replaced with the framework sequence of the germline IGHV3-23 using the CDR transplantation method, and the chimeric antibody was labeled Hu2117-H1; the light chain framework sequence of mAb2117 was replaced with the framework sequence of the germline IGKV1-39, and the chimeric antibody was labeled Hu2117-K1. The mAb2117 chimeric antibody and its humanized antibody were respectively constructed into single-chain antibody fusion proteins of hIgG1Fc (scFv-hIgG1Fc fusion proteins), and the scFv antibody gene fragments were obtained by gene sequence synthesis, namely the humanized single-chain antibodies Hu2117HK and Hu2117KH and the chimeric single-chain antibody controls Mu2117HK and Mu2117KH, wherein HK represents the structure of the single-chain antibody as "(N-terminus) heavy chain variable region nucleic acid sequence-linker (G4S)3-light chain variable region nucleic acid sequence (C-terminus)", and KH represents the structure of the single-chain antibody as "(N-terminus) light chain variable region nucleic acid sequence-linker (G4S)3-heavy chain variable region nucleic acid sequence (C-terminus)". The scFv gene fragments were homologously recombined into the pcDNA3.1 (containing human IgG1Fc sequence) vector to construct antibody expression plasmids. After the expression plasmids were extracted, they were transiently transferred into ExpiCHO-S cells (Thermo) for serum-free culture. The antibodies in the culture supernatant were purified by Protein A affinity chromatography using the AKTA Pure system to obtain single-chain antibody fusion proteins. The binding kinetics of the single-chain antibody fusion protein and the HER2 recombinant protein were analyzed using ForteBio. The test results are shown in Table 5. The humanized antibody basically retained the binding activity with HER2, and the affinity constant (K D ) is about 10 -7 M.

[0373] Table 5. Binding kinetics analysis results of single-chain antibody fusion protein and HER2 recombinant protein Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0374] According to the method described in item 1.5 of Example 1, the binding activity of the single-chain antibody fusion proteins Hu2117HK and Hu2117KH to BT474 cells was detected by flow cytometry, where the chimeric antibodies mAb2117 and Trastuzumab were used as positive controls. The results showed that mAb2117, Hu2117HK, and Hu2117KH could all bind to BT474 cells, among which the activity of Hu2117HK in binding to BT474 cells was slightly higher than that of Hu2117KH (data not shown).

[0375] 2.2 Single-point or multi-point mutation optimization of the anti-HER2 humanized antibody Hu2117HK

[0376] Using the online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, we analyzed amino acid residues with low humanization levels, potential stability risk sites, and murine amino acid residues in the framework regions of the light and heavy chain variable regions of the humanized antibody Hu2117HK. A total of 50 single-point / multi-point mutations were designed to improve the antibody's degree of humanization, antigen-binding affinity, and / or stability. Specifically, these 50 designed single-point / multi-point mutations were introduced into the single-chain antibody Hu2117HK sequence via PCR, and the single-chain antibody mutant proteins were expressed.

[0377] The ForteBio molecular interaction instrument was used to detect the transient expression levels of the above 50 single-chain antibody mutants and their parent single-chain antibody Hu2117HK. The specific method is as follows: After transient expression in ExpiCHO-S cells, the culture supernatant was harvested and added to a 96-well black plate at 200 μL / well. The test sample and Protein A probe were placed in the ForteBio instrument, and the binding rate of each antibody molecule in the culture supernatant to the Protein A probe was detected using a preset quantitative program; after each cycle, the probe was immersed in 10mM glycine buffer (pH 1.5) to regenerate the probe, and then the next cycle of detection was started. After the detection was completed, the expression level of each single-chain antibody was calculated using the standard curve using analysis software. At the same time, the binding kinetics of each single-chain antibody mutant to HER2 were analyzed using ForteBio according to the method described in Item 1.8 of Example 1.

[0378] The results of single-point / multi-point mutation sites and mutated amino acids, transient expression levels, and antigen binding kinetic analysis are shown in Table 6.

[0379] Table 6. Single-point / multi-point mutations, transient expression levels, and HER2 binding kinetics analysis results of the single-chain antibody Hu2117HK L is light chain; H is heavy chain; N / A means not applicable; Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0380] According to the method described in item 1.5 of Example 1, the binding activity of the single-chain antibody containing single / multi-point mutations to BT474 cells and RT-112 cells was detected by flow cytometry, wherein the positive control antibody was the parent single-chain antibody Hu2117HK. The results are shown in Figure 7. Compared with the parent single-chain antibody Hu2117HK, most of the single-chain antibody mutants retained the binding activity to BT474 cells, and the cell binding activity of some antibodies was even slightly improved. These antibodies include Hu2117HK-Mu01, -Mu02, -Mu03, -Mu04, -Mu05, -Mu06, -Mu08, -Mu09, -Mu10, -Mu11, -Mu12, -Mu13, -Mu17, -Mu20, -Mu21, -Mu23, -Mu24, -Mu25, -Mu27, -Mu28, -Mu32, -Mu33, -Mu36, -Mu37, -Mu38, -Mu39, -Mu40, -Mu45, -Mu46, -Mu47, -Mu48, -Mu49, and -Mu50 (Figure 7A). At the same time, the binding results to RT-112 cells are shown in Figure 7B. Compared with the parent single-chain antibody Hu2117HK, most of the single-chain antibody mutants retained the binding activity to RT-112 cells. These antibodies include Hu2117HK-Mu01, -Mu02, -Mu03, -Mu04, -Mu05, -Mu06, -Mu08, -Mu09, -Mu10, -Mu11, -Mu12, -Mu13, -Mu14, -Mu23, -Mu24, -Mu25, -Mu27, -Mu28, -Mu29, -Mu32, -Mu34, -Mu37, -Mu38, -Mu39, -Mu40, -Mu41, -Mu42, -Mu45, -Mu46, -Mu48, -Mu49, and -Mu50.

[0381] 2.3 Combination mutation optimization of the anti-HER2 humanized antibody Hu2117HK

[0382] The above-mentioned preferred single-point / multi-point mutations were combined and introduced into the variable region sequence of the humanized single-chain antibody Hu2117HK to construct antibody molecules containing different combinations of mutations Hu2117-HK201 to -HK203 (abbreviated as HK201 to HK203), and Hu2117-HK301 to -HK312 (abbreviated as HK301 to HK312). Single-chain antibody samples were prepared according to the method described in Item 2.2 of this Example, and the transient expression levels of each mutant and the parent single-chain antibody Hu2117HK were detected using ForteBio. The combined mutations and transient expression levels contained in each antibody molecule are shown in Table 7.

[0383] Table 7. Combination mutation optimization and transient expression of single-chain antibody Hu2117HK

[0384] Flow cytometry was used to test the binding activity of the single-chain antibodies containing combined mutations and their parent single-chain antibody, Hu2117HK, to BT474 and RT-112 cells, respectively, according to the method described in Item 1.5 of Example 1. The results showed that at a saturating concentration of 10 μg / mL, antibodies HK201, HK202, and HK203 were able to bind to both BT474 and RT-112 cells, with binding activities comparable to those of the parent antibody, Hu2117HK. Furthermore, antibodies HK301, HK303, HK304, HK305, HK306, HK309, and HK310 were able to bind to RT-112 cells, with binding activities comparable to those of the parent antibody, Hu2117HK (data not shown).

[0385] To further characterize the cell-binding activity of the antibodies optimized through combinatorial mutations, preferably the HK203, HK303, HK304, HK309, and HK310 antibodies, the antibodies were serially diluted and their binding to BT474 and RT-112 cells was assayed by flow cytometry according to the method described in Section 1.5 of Example 1. The control antibody was the parental single-chain antibody Hu2117HK. The results, shown in Table 8 and Figure 8, show that these optimized humanized antibodies all bound to BT474 and RT-112 cells with binding activity comparable to that of the parental antibody Hu2117HK.

[0386] Table 8. Binding activity of the single-chain antibody Hu2117HK combined mutation-optimized molecules to BT474 cells and RT-112 cells

[0387] Example 3 Humanization and Optimization of Anti-HER2 Chimeric Antibody mAb2126

[0388] 3.1 Humanization of the anti-HER2 chimeric antibody mAb2126

[0389] After analysis by MOE software, the heavy chain framework sequence of the chimeric antibody mAb2126 was replaced with the framework sequence of germline IGHV4-4 or IGHV7-4 using the CDR grafting method, and they are labeled as Hu2126-H1 and Hu2126-H2 respectively; the light chain framework sequence of mAb2126 was replaced with the framework sequence of germline IGKV1-39, and they are labeled as Hu2126-K1. Humanized antibody variable region gene fragments were obtained by gene sequence synthesis. The light and heavy chain gene fragments were respectively ligated with the linearized expression vector pcDNA3.1 to construct antibody expression plasmids (the heavy chain variable region [VH] was ligated with the pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region [VL] was ligated with the pcDNA3.1 vector containing the human CK constant region). The expression plasmids were extracted according to conventional methods and transiently transferred into ExpiCHO-S cells (Thermo) for serum-free expression culture. The culture supernatant was purified by Protein A affinity chromatography using the AKTA Pure system to obtain humanized antibodies Hu2126-H1K1 and Hu2126-H2K1.

[0390] The binding kinetics of each humanized antibody to HER2 were tested using ForteBio according to the method described in Item 1.8 of Example 1. The results are shown in Table 9. The humanized antibodies obtained by CDR grafting retained HER2 binding activity, with the binding affinity of Hu2126-H2K1 being comparable to that of the parent chimeric antibody mAb2126.

[0391] Table 9. Binding kinetic analysis results of chimeric antibody mAb2126 before and after humanization (CDR grafting)

[0392] Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0393] 3.2 Single-point mutation optimization of the anti-HER2 humanized antibody Hu2126-H2K1

[0394] After analyzing the amino acid residues with relatively low humanization degree, potential molecular stability risk sites, and mouse amino acid residues in the framework region of the light and heavy chain variable region sequences of the humanized antibody Hu2126-H2K1 using the online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, a total of 63 single point mutations were designed to improve the degree of humanization, antigen binding affinity, and / or stability of the antibody. Specifically, the 63 designed single point mutations were introduced into the humanized antibody Hu2126-H2K1 sequence by PCR, and expression plasmids were constructed. The antibody light and heavy chain expression plasmids were then combined and transiently transfected into ExpiCHO-S cells for antibody expression, obtaining 63 antibody mutant expression supernatants containing single point mutations. According to the method described in Example 2, Item 2.2, the expression level of each antibody mutant after 3 days of transient culture and the binding kinetics to HER2 were detected using ForteBio.

[0395] The results are shown in Table 10. Compared with the parent antibody Hu2126-H2K1, a total of 42 of the 63 designed single point mutations did not significantly affect the antigen binding affinity of the antibody, including 17 single point mutations in the antibody light chain: 2126-Hu-L02, -L05, -L06, -L07, -L08, -L09, -L10, -L11, -L12, -L13, -L14, -L15, -L16, -L17, -L18 , -L19, -L21, and 25 single point mutations in the antibody heavy chain: 2126-Hu-H22, -H23, -H24, -H25, -H27, -H28, -H29, -H31, -H32, -H33, -H34, -H35, -H36, -H37, -H38, -H39, -H40, -H42, -H43, -H44, -H45, -H46, -H50, -H54, -H59.

[0396] Table 10. Single point mutations, transient expression levels, and HER2 binding kinetics analysis results of Hu2126-H2K1 humanized antibody L is light chain; H is heavy chain; N / A stands for not applicable; Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0397] 3.3 Combination mutation optimization of the anti-HER2 humanized antibody Hu2126-H2K1

[0398] Preferably, the above-mentioned single point mutations in the light chain are combined and introduced into Hu2126-K1, and preferably, the above-mentioned single point mutations in the heavy chain are combined and introduced into Hu2126-H2. Light chain mutation combination expression plasmids and heavy chain mutation combination expression plasmids are constructed, respectively. The light and heavy chain expression plasmids are then paired and transiently transfected into ExpiCHO-S cells. After culture, expression, and purification, Hu2126-H2K1-L71-H72b-Mu10 to -Mu19 (abbreviated as 7172b-Mu10 to -Mu19) are obtained, for a total of 10 antibody mutants. The binding kinetics of the humanized antibodies containing the combined mutations to HER2 are tested using ForteBio according to the method described in Item 1.8 of Example 1.

[0399] The combined mutations contained in each antibody mutant and the results of antigen binding kinetic analysis are shown in Table 11. The binding affinity of antibodies 7172b-Mu10, 7172b-Mu14, 7172b-Mu17 and 7172b-Mu19 to HER2 is significantly higher than that of the parent antibody Hu2126-H2K1.

[0400] Table 11. Combination mutation optimization of antibody Hu2126-H2K1 and analysis of binding kinetics with HER2 Full R 2 Indicates the similarity between the fitted curve and the measured curve.

[0401] Example 4 Construction, Optimization and Functional Activity Identification of Anti-HER2 Complementary Bispecific Antibodies

[0402] 4.1 Construction of complementary anti-HER2 bispecific antibodies

[0403] In this example, a pair of monospecific antibodies (Hu2117-HK304 and Hu2126-H2K1-L71-H72b-Mu14) that can respectively recognize different epitopes in the extracellular region of HER2 and do not compete with each other were selected to construct an exemplary complementary bispecific antibody. As shown in Figure 9, the bispecific antibody configuration is a DVD-IgG structure, the Fv domain is the variable region sequence of Hu2117-HK304, and the IgG domain is the Hu2126-H2K1-L71-H72b-Mu14 sequence, wherein the heavy chain variable region of the Fv domain is connected to the heavy chain of the IgG domain through a linker (G4S) 1, and the light chain variable region of the Fv domain is connected to the light chain of the IgG domain through a linker (G4S) 3. At the same time, in order to avoid the effector function mediated by the antibody Fc region from causing killing effects on non-target cells, the L234F / L235E / P331S (EU Numbering) mutation (referred to as "TM mutation") is introduced into the Fc region of the bispecific antibody molecule to reduce its binding to FcγRs and C1q, thereby eliminating the Fc effector function. The constructed complementary bispecific antibody is named 04BS-109-WT. The 04BS-109-WT bispecific antibody expression plasmid was transiently transfected into ExpiCHO-S cells for antibody protein expression and purification by Protein A affinity chromatography. During the purification process, it was found that the 04BS-109-WT bispecific antibody molecule formed a colloidal precipitate during elution (the eluent did not contain NaCl), and the eluted product appeared turbid in the solution. However, after the addition of 0.1 M NaCl, the colloidal precipitate gradually dissolved, and the antibody solution eventually became clear.

[0404] 4.2 Optimization of complementary anti-HER2 bispecific antibodies

[0405] Using MOE software analysis, a series of mutations were designed within the HCDR2 sequence of the Fv domain of the complementary bispecific antibody 04BS-109-WT to improve the stability of the bispecific antibody. These designed mutations are shown in Table 12 and are designated 04BS-1123-ST01 through -ST06. Mutations were introduced into the 04BS-109-WT sequence via PCR. The constructed bispecific mutant expression plasmids were transiently transfected into ExpiCHO-S cells and cultured in a serum-free culture to express the antibody proteins. Antibody expression in the culture supernatant was measured using ForteBio as described in Example 2, Section 2.2. The results showed that the expression level of the 04BS-1123-ST03 antibody was too low (<30 mg / L), while the expression levels of the remaining bispecific mutants were all above 300 mg / L after 10 days of transient culture. After purification by Protein A affinity chromatography, SEC-HPLC analysis revealed that the monomer purity of each bispecific mutant was close to 100%. However, significant differences in molecular stability were observed among the bispecific antibody mutants during purification (as shown in Table 12). During the Protein A affinity chromatography purification and elution process, the sample solution of the 04BS-1123-ST06 bispecific antibody remained clear, regardless of whether the eluent contained 0.1 M NaCl. This suggests that the molecular stability of the 04BS-1123-ST06 bispecific antibody appears to be superior to that of the other mutants and the parent antibody 04BS-109-WT.

[0406] Table 12. Molecular stability performance of exemplary anti-HER2 complementary bispecific antibody 04BS-109-WT and its mutants Note: The underlined amino acid residues represent the amino acid mutations introduced into the HCDR2 sequence of Hu2117-HK304.

[0407] 4.3 Cell competition binding assay of anti-HER2 humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14

[0408] The anti-HER2 monospecific antibody derived from the Fv domain sequence of 04BS-1123-ST06 was named Hu2117-HK304-06. To further confirm whether the optimized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 retained the property of their parent antibodies (chimeric antibodies mAb2117 and mAb2126) of not competing for HER2 binding, this experiment followed the method described in Example 1, Section 1.5. These two optimized antibodies, serially diluted from 800 μg / mL, were mixed with 8 μg / mL biotin-labeled Hu2117-HK304-06 antibody in equal volumes and incubated with BT474 cells. AF488-labeled streptavidin (Jackson Immuno) was then used as a secondary antibody to assess the competitive inhibitory activity of these two antibodies against HER2-expressing tumor cells by flow cytometry. The results are shown in FIG10 . Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 do not compete for binding to HER2 expressed on the cell surface.

[0409] 4.4 Analysis of the endocytic response and lysosomal transport / degradation efficiency of anti-HER2 complementary bispecific antibodies

[0410] In this example, flow cytometry was used to measure the endocytosis rate of the exemplary anti-HER2 complementary bispecific antibody in cells expressing different levels of HER2 (including BT474, JIMT-1, and RT-112). The specific method is as follows: After the cells were digested with trypsin, they were washed with pre-cooled FACS buffer and resuspended to 6-10×10 6cells / mL; the cell suspension was transferred to a 96-well U-shaped deep-well plate at 300 μL / well, and then 300 μL of the test antibody was added to each well (the final concentration of the antibody was 20 μg / mL), mixed and incubated on ice for 30 to 60 minutes; after washing twice with pre-cooled FACS buffer, the cells in each well were fully resuspended with 600 μL / well pre-cooled FACS buffer, and then the cell suspension was evenly distributed into 5 96-well U-shaped plates at 100 μL / well, one of the plates was randomly selected as the control group and incubated on ice, and the other 4 plates were selected as the experimental groups and incubated on ice for 3 minutes. Incubate at 7°C and transfer one of the plates to ice after 30 minutes, 1 hour, 2 hours, and 4 hours to terminate the endocytosis reaction; after incubation of all wells, add pre-cooled FACS buffer to each well to wash the cells twice, add 100 μL / well fixative (2% paraformaldehyde), and place at 4°C overnight; add pre-cooled FACS buffer to each plate and wash twice, then add 100 μL / well AF488-labeled anti-human IgG (H+L) antibody (Jackson Immuno) diluted 1:1000 in pre-cooled FACS buffer, resuspend the cells and incubate on ice in the dark for 40 minutes; add pre-cooled FACS buffer to wash the cells twice, resuspend the cells in 100 μL / well FACS buffer and detect them using flow cytometry. The internalization rate (percentage value) of the antibody is calculated as follows: (MFI 对照组 -MFI 实验组 ) / MFI 对照组 × 100%. The results are shown in Figure 11. Compared with the control antibody trastuzumab and the related monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14, the anti-HER2 complementary bispecific antibodies 04BS-1123-ST04, 04BS-1123-ST05, 04BS-1123-ST06 and their parent antibody 04BS-109-WT can induce more rapid and efficient endocytosis in BT474, JIMT-1 and RT-112 cells. Among them, the endocytosis rate of the complementary bispecific antibodies in JIMT-1 and RT-112 cells can reach about 60%, and the endocytosis rate in BT474 cells can reach as high as about 80%.

[0411] To further verify that the anti-HER2 complementary bispecific antibody was transported into the lysosome after endocytosis, the subcellular localization of the antibody was detected using confocal microscopy in this example. The specific method is as follows: SKBR-3 cells were collected and plated onto five 8-well chamber slides (Thermo) for culture; after the cells grew into a uniform monolayer, the culture medium was discarded and the cells were rinsed once with PBS; 500 μL of 10 μg / mL anti-HER2 complementary bispecific antibody 04BS-1123-ST06, control antibody trastuzumab, or human IgG isotype control antibody (such as the anti-HIV / gp120 antibody described in WO2003106496A1) was added to each chamber of the chamber slide and incubated on ice for 1 hour; one of the chamber slides was selected as a 0-hour control, rinsed once with pre-cooled PBS, and fixed with 2% paraformaldehyde; the remaining four slides were incubated at 37°C for 0.5 hours, 2 hours, 4 hours, and 8 hours, rinsed once with pre-cooled PBS, and fixed with 2% paraformaldehyde at 4°C overnight; PBST (PBS containing 0.05% After rinsing twice with Tween-20, 250 μL of fixation and permeabilization buffer (PBS containing 2% goat serum and 0.5% Triton X-100) was added to each chamber and incubated at room temperature for 10 minutes; after rinsing twice with PBST, 250 μL of mouse anti-LAMP1 antibody (1:50 dilution, BD) and AF488-labeled goat anti-human IgG antibody (2 μg, Molecular Probes) diluted in FACS buffer (PBS containing 2% FBS) were added to each chamber and incubated at room temperature for 1 hour in the dark; after rinsing twice with PBST, 250 μL of AF647-labeled goat anti-mouse IgG antibody (1:250 dilution, Molecular Probes) were added to each chamber. The cells were incubated in FACS buffer (1 μg / mL) at room temperature in the dark for 1 hour; rinsed twice with PBST, and stained with DAPI at a final concentration of 1 μg / mL for 1 minute in each chamber; rinsed once with PBST, and sealed with sealing solution (Thermo) and incubated at room temperature in the dark overnight; scanned and analyzed using a confocal microscope.The results are shown in Figure 12. No green fluorescence signal was detected in cells incubated with the human IgG isotype control antibody at any time point, indicating that there was no non-specific antibody binding to the cells. Green fluorescence signals were detected in cells incubated with the positive control antibody Trastuzumab, and there was no significant difference at any time point. The green fluorescence signals were evenly distributed in a ring shape on the cell membrane and did not overlap with the lysosomes labeled with red fluorescence, indicating that Trastuzumab was able to bind to HER2 on SKBR-3 cells, but no significant endocytosis and lysosomal transport occurred. In contrast, in cells incubated with the anti-HER2 complementary bispecific antibody 04BS-1123-ST06, as the treatment time progressed, the green fluorescence signal gradually shifted from the cell membrane to the cell and overlapped with the red fluorescence-labeled lysosomes (yellow, as indicated by the arrow in the figure). Thereafter, the intensity of the yellow fluorescence signal gradually weakened or even disappeared completely over time, indicating that the internalized anti-HER2 complementary bispecific antibody 04BS-1123-ST06 was transported into the lysosomes and degraded, and its lysosomal transport efficiency was almost 100%.

[0412] The degradation of HER2 was detected by Western blot, which further verified that the anti-HER2 complementary bispecific antibody was internalized and transported into lysosomes for degradation after binding to HER2 on the cell surface. The specific method is as follows: BT474 cells were collected and resuspended in complete culture medium (RPMI-1640 medium containing 10% FBS) to 1×10 6cells / mL, and the cell suspension was dispensed into 1.5-mL EP tubes at 500 μL / tube; the antibodies to be tested (including the complementary bispecific antibody 04BS-1123-ST06, the control antibody Trastuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 corresponding to the complementary bispecific antibodies) were diluted to 300 μg / mL with complete culture medium, and then 100 μL / tube was added to the above EP tubes (the final antibody concentration was 50 μg / mL); each EP tube was placed at 37°C / 5% The cells were incubated in a CO2 incubator for 24 hours, then centrifuged and washed twice with pre-chilled PBS; RIPA lysis buffer (Proteintech) was added to 100 μL / tube to resuspend the cells and incubated on ice for 30 minutes; each tube was centrifuged at 4°C for 10 minutes, the supernatant of the lysate was collected, and the protein concentration in the extract was quantified using a BCA kit (Thermo); based on the protein concentration, the protein concentration in each extract was diluted to 0.125 μg / μL with RIPA lysis buffer, and then 5× SDS-PAGE sample buffer containing DTT was added and heated at 95°C for 5 minutes to prepare SDS-PAGE loading samples. The treated samples were loaded onto a 10% SDS-PAGE gel at 10 μL / lane (i.e., 1 μg / lane) for electrophoresis, and the proteins were then transferred to a PVDF membrane at 80 V. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour, and then the membrane was cut in half at the 55 kDa molecular weight marker. The upper half was added with a 1:5000 dilution of rabbit anti-human HER2 antibody (CST) and incubated at 4°C overnight, and the lower half was added with a 1:10,000 dilution of mouse anti-human GAPDH antibody (Proteintech) and incubated at room temperature for 1 hour. After incubation, the membrane was washed three times with PBST, and then goat anti-rabbit IgG-HRP antibody (Jackson Immuno) and goat anti-mouse IgG-HRP antibody (Jackson Immuno) were added at 1:5000 dilutions respectively and incubated at room temperature for 1 hour. The membrane was washed three times with PBST and then exposed to light using ECL developer (NanoVizon). The results are shown in FIG13 . The complementary bispecific antibody 04BS-1123-ST06 can effectively induce the degradation of HER2 in BT474 cells, while the control antibody cannot induce the degradation of HER2.

[0413] This example also examined the effect of the complementary bispecific antibody 04BS-1123-ST06 on BT474 cell proliferation. The specific method was as follows: BT474 cells were seeded in 96-well white-walled plates (10,000 cells / well) and serially diluted test antibodies (including 04BS-1123-ST06, trastuzumab, pertuzumab, Hu2117-HK304-06, and Hu2126-H2K1-L71-H72b-Mu14) were added. The cells were incubated at 37°C / 5% CO2 for 5 days before the detection reagent CellCounting-Lite 2.0 Luminescent Reagent (Nanjing Novozymes) was added. Chemiluminescence values ​​were read using a multi-function microplate reader (Varioskan, Thermo). The experimental data were analyzed using GraphPad Prism 9 software. The results, as shown in Figure 14, show that the complementary bispecific antibody 04BS-1123-ST06 effectively inhibited the proliferation of BT474 cells, whereas none of the corresponding monospecific antibodies showed any inhibitory activity against BT474 cell proliferation. Since the proliferation of BT474 cells relies on signal transduction mechanisms activated by the formation of homodimers of overexpressed HER2 on the cell membrane, it is inferred that the cell proliferation inhibitory activity of the complementary bispecific antibody is due to the endocytosis / degradation of cell surface HER2, which prevents the formation of HER2 homodimers.

[0414] 4.5 Effects of anti-HER2 complementary bispecific antibodies on NRG-1-induced HER2 / HER4 dimerization

[0415] Promega's NanoBiT structural complementation reporter system was used to further investigate whether anti-HER2 complementary bispecific antibodies could affect NRG-1-induced HER2 / HER4 dimerization. The NanoBiT system consists of an LgBiT subunit and a SmBiT subunit, each of which can be fused to a test protein. If the two test proteins interact to form a dimer, LgBiT complements the SmBiT structure to form a functional luciferase, which then reacts with the substrate to produce a luminescent signal. In this example, DNA sequences encoding the extracellular and transmembrane regions of HER2 and HER4 were first inserted into the NanoBiT system pBiT1.3-C and pBiT2.3-C vectors using molecular cloning techniques. These two plasmids were then co-transfected into U2OS cells. Cells were harvested 24 hours after transfection, plated in 96-well white-walled plates, and cultured overnight at 37°C. Complementary bispecific antibody 04BS-1123-ST06, humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14, chimeric antibodies mAb2117 and mAb2126, and control antibodies trastuzumab and pertuzumab were serially diluted and mixed with NRG-1 at a 1:1 volume ratio (final NRG-1 concentration of 1 nM). Nano-Glo Live Cell Reagent (Promega) was added to a 96-well white-walled plate, and the chemiluminescence value was measured as a background. Subsequently, the prepared samples were added to each well and incubated at 37°C for 6 minutes. Chemiluminescence values ​​were then measured and analyzed using GraphPad Prism 9 software. The results are shown in Figure 15. The control antibodies Trastuzumab and Pertuzumab can significantly inhibit NRG-1-induced HER2 / HER4 dimerization, while the chimeric antibodies mAb2117 and mAb2126, the monospecific humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (Figure 15A), and the complementary bispecific antibody 04BS-1123-ST06 (Figure 15B) constructed based on these two monospecific humanized antibodies have no inhibitory effect. This result further illustrates that the anti-HER2 complementary bispecific antibody does not affect the normal biological function of HER2, and therefore its risk of inducing cardiac toxicity in vivo is extremely low. 4.6 Effect of anti-HER2 complementary bispecific antibodies on NRG-1-induced activation of HER2 downstream signal transduction pathways

[0416] Human induced pluripotent stem cells (iPSC) and related culture kits were purchased from Fujiflim Cellular Dynamics. According to the method described in the instructions provided by the manufacturer, iPSCs were plated into 6-well plates and cultured at 37°C and 5% CO2 for one week to allow the cells to fully differentiate into human cardiomyocytes (iCell Cardiomyocytes). The effect of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes was detected according to the method described in Item 1.10 of Example 1. As shown in Figure 16, unlike the positive control antibody Pertuzumab, 04BS-1123-ST06 had no inhibitory effect on NRG-1-induced AKT phosphorylation in human cardiomyocytes, indicating that the complementary bispecific antibody does not affect the regulation of NRG-1-induced HER2 dimerization and its downstream signal transduction pathways in cardiomyocytes.

[0417] 4.7 Fc effector function modification of anti-HER2 complementary bispecific antibodies

[0418] To mitigate potential safety risks associated with Fc effector function, this example introduces three point mutations in the Fc region of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06: L234F / L235E / P331S (TM mutations). These mutations reduce the antibody's binding activity to Fc receptors and C1q. ForteBio assays confirmed that the complementary bispecific antibody 04BS-1123-ST06, containing the TM mutations, showed no significant binding to FcγRI, FcγRIIa (167H), FcγRIIb, FcγRIIIa (176V), FcγRIIIa (176F), and C1q. However, the binding affinity of the complementary bispecific antibody to FcRn was not significantly altered compared to that of trastuzumab (data not shown).

[0419] In addition, this example further verified whether the complementary bispecific antibody 04BS-1123-ST06 containing the TM mutation had ADCC activity, using Jurkat cells (Promega) expressing the FcγRIIIa-176V receptor and a firefly luciferase reporter gene regulated by the NFAT response element as effector cells, BT474 cells overexpressing HER2 as target cells, and trastuzumab as a positive control antibody. The specific method was as follows: BT474 cells were collected by centrifugation, resuspended in ADCC assay buffer (RPMI-1640 medium containing 0.5% FBS), and then diluted to 1.5×10 4Add 10 cells / well to a 96-well white plate. Dilute the antibody to be tested with ADCC assay buffer in a gradient manner and add 25 μL / well to the above 96-well plate. Collect the effector cells by centrifugation and resuspend them in ADCC assay buffer at a concentration of 1.5×10 5 Cells / well were added to the above-mentioned 96-well plate and then incubated overnight at 37°C and 5% CO2. The next day, the 96-well plate was equilibrated at room temperature, and then the detection reagent Bio-Lite Luciferase Reagent (Nanjing Novozyme) was added. The chemiluminescence value was read using a multifunctional microplate reader (Varioskan, Thermo), and the experimental data were analyzed using GraphPad Prism 9 software. The test results are shown in Figure 17. Unlike the positive control antibody Trastuzumab, 04BS-1123-ST06 did not induce any luciferase expression even at the highest concentration, indicating that the anti-HER2 complementary bispecific antibody with the TM mutation introduced in the Fc region does not have ADCC activity.

[0420] Example 5 Preparation and functional activity identification of anti-HER2 complementary bispecific antibody ADC

[0421] 5.1 Preparation of Anti-HER2 Complementary Bispecific Antibody-Drug Conjugates

[0422] In this example, the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 was conjugated to the small molecule toxin compound eribulin with anti-tumor activity via a cleavable linker (e.g., Mal-(PEG)2-VC-PAB, Mal-(PEG)2-GGFG) to prepare an anti-HER2 complementary bispecific ADC. Eribulin was prepared according to the methods described in ZL201910197071.8 or ZL201910509222.9, and WO1999065894.

[0423] The preparation of the drug payload (Payload) Mal-(PEG)2-VC-PAB-Eribulin was carried out according to the method described in Example 2.1.1 of WO2017151979.

[0424] The drug payload, Mal-(PEG)2-GGFG-Eribulin, was prepared as follows: NH2-(PEG)2-COOH and maleic anhydride were added to acetic acid, heated under reflux overnight, and the acetic acid was removed by rotary evaporation. The residue was purified by HPLC and lyophilized to obtain Mal-(PEG)2-COOH. Using 2Cl Trt Resin as a solid support, the Fmoc protection was removed using a 20% piperidine / DMF (v / v) solution. Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Gly-OH were then coupled sequentially using HOBT / DIC as a condensation system and DMF as the reaction solvent. The product was then cleaved using a DCM:TFE:AcOH (7:2:1) solution, precipitated with methyl tert-butyl ether, centrifuged, dried, and purified by HPLC to obtain Fmoc-GGFG-OH. Fmoc-GGFG-OH and eribulin were placed in a single-necked flask, dissolved in DMF, cooled to 0°C, and then DIPEA (161.3 mg, 1.25 mmol) and DECP (122.3 mg, 0.75 mmol) were added. The mixture was allowed to react at room temperature and monitored by HPLC. After completion, the reaction solution was poured into MTBE (100 mL), stirred at room temperature for 1 hour, filtered, and the filter cake washed with MTBE to obtain crude Fmoc-GGFG-eribulin. The crude Fmoc-GGFG-eribulin was placed in a single-necked flask, added with THF, cooled to 0°C, and then added with two equivalents of aqueous LiOH solution. The mixture was allowed to react at room temperature and monitored by HPLC. After completion, the pH was adjusted to approximately 6-7 with 50% acetic acid, and the THF was removed by rotary evaporation. The mixture was then purified by HPLC to collect pure NH2-GGFG-eribulin. HATU was added to a DMF solution of Mal-(PEG)2-COOH and sodium bicarbonate at room temperature. After stirring for 30 minutes, an equivalent amount of free NH2-GGFG-Eribulin was added to the above solution and stirred for another hour until the reaction was complete as determined by TLC. The solid was removed by filtration, and the filtrate was purified by HPLC to obtain Mal-(PEG)2-GGFG-Eribulin.

[0425] The preparation method of the anti-HER2 complementary bispecific ADC is as follows: ZnCl2 (3.0mM) and TCEP (6.0mM) are slowly added to a solution of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 (1.5mM) (the antibody preparation buffer is 50mM Histidine-HCl, 8% sucrose, pH 5.5) under an ice-water bath. The final reaction concentrations of ZnCl2, TCEP and 04BS-1123-ST06 antibody are 0.10mM, 0.20mM and 0.05mM, respectively. After mixing, the mixture is reduced by shaking at 8°C for approximately 16 hours. Mal-(PEG)2-VC-PAB-Eribulin or Mal-(PEG)2-GGFG-Eribulin dissolved in DMSO was added to a final concentration of 0.32 mM under ice water bath. After the reaction was continued at 8°C for 3 hours, cysteine ​​was added to a final concentration of 0.05 mM to deplete the excess Mal-(PEG)2-VC-PAB-Eribulin or Mal-(PEG)2-GGFG-Eribulin. EDTA was added to a final concentration of 0.15 mM to chelate Zn 2+ , using Pellicon XL Ultrafiltration membrane filtration package (Module Ultracel 30kDa / 0.005m 2 , purchased from Merck) to remove related impurities. Finally, HIC-HPLC (BioCore HIC-Butyl 5μm / 4.6×100mm, purchased from NanoChrom) was used to analyze the drug-antibody ratio (DAR) and heterogeneity of the prepared ADC, and SEC-HPLC (Zenix-C SEC-300, purchased from Saifen Technology) was used to analyze the purity of the ADC product.

[0426] The average DAR value of the prepared ADC can be calculated by the following formula: Average DAR value = [AUC +1 +2(AUC +2 )+3(AUC +3 )+...+n(AUC +n )] / ΣAUC 总 ]. Among them, AUC +1 AUC is the area under the peak curve corresponding to the ADC coupled with a small molecule compound. +2 is the area under the peak curve corresponding to the ADC coupled with two small molecule compounds, and so on, ΣAUC 总 The average DAR value of the ADC prepared in this example was 4.0±0.5, and the purity was >95%.

[0427] 5.2 Validation of Antigen Binding Specificity of Anti-HER2 Complementary Bispecific ADCs

[0428] According to the method described in Item 1.4 of Example 1, the binding activity of the exemplary anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) to the HER2 recombinant protein and the cross-binding activity with other members of the human ErbB / HER family were detected by ELISA. The antigens included the human HER2 extracellular domain recombinant protein and the human EGFR, HER3, and HER4 extracellular domain recombinant proteins. The test results are shown in Figure 18. The anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) can specifically bind to HER2, but has no cross-binding activity with other members of the human ErbB / HER family. The antigen binding specificity of the anti-HER2 complementary bispecific ADC is comparable to that of its corresponding naked antibody 04BS-1123-ST06 (data not shown), indicating that the antigen binding specificity of the complementary bispecific antibody is not changed after coupling with the small molecule toxin compound.

[0429] 5.3 Detection of in vitro tumor cell killing activity of anti-HER2 complementary bispecific ADCs

[0430] In this example, a series of tumor cell lines expressing different levels of HER2 were selected to evaluate the in vitro cell killing activity of the anti-HER2 complementary bispecific ADC. The HER2 expression levels and drug resistance of the tumor cell lines used are shown in Table 13; among them, JIMT-1 is a cell line that overexpresses HER2 and is congenitally resistant to DS-8201 (in vitro activity), and SKOV-3R* is a cell line that overexpresses HER2 and is acquiredly resistant to DS-8201.

[0431] The specific steps for in vitro tumor cell cytotoxicity assays are as follows: Cells were harvested by trypsinization, resuspended in medium containing 10% FBS, and seeded into 96-well white-walled plates at a density of 5,000 or 10,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After complete attachment, ST06-VCP-Eribulin, ST06-GGFG-Eribulin, or DS-8201 diluted in the corresponding culture medium were added. Depending on the cell growth rate, the plates were incubated at 37°C / 5% CO2 for 3–5 days. After equilibration to room temperature, CellCount Lite 2.0 reagent (NanoVegas, Nanjing) was added and shaken for 2–5 minutes to fully lyse the cells. After incubation at room temperature for 10 minutes, chemiluminescence was measured using a multi-function microplate reader (Varioskan, Thermo). Data were analyzed using GraphPad Prism 9 software and expressed as a percentage of the chemiluminescence value relative to the untreated control wells.

[0432] The test results are shown in Figure 19 and Table 13. In the HER2-overexpressing tumor cell lines NCI-N87, SKBR-3, and SKOV-3, the anti-HER2 complementary bispecific ADCs (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) and the benchmark molecule DS-8201 all exhibited strong cytotoxicity, and the cell-killing activity of the complementary bispecific ADCs was significantly higher than that of DS-8201. In the HER2-low-expressing tumor cell lines MDA-MB-361 and MDA-MB-453, the cell-killing activity of the complementary bispecific ADCs was significantly higher than that of DS-8201. In the HER2-low-expressing tumor cell line ZR-75-1, the complementary bispecific ADCs also showed effective cell-killing activity, while DS-8201 had almost no cell-killing activity. In tumor cell lines T47D, RT-112, and MCF-7 that express lower levels of HER2, as well as in the tumor cell line MDA-MB-468 that does not express HER2, the complementary bispecific ADC showed no significant cytotoxicity, indicating that the complementary bispecific ADC has a good safety window. In addition, in JIMT-1 cells that highly express HER2 and have innate / intrinsic resistance to DS-8201, the complementary bispecific ADC (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) also showed significant cytotoxicity. Thus, the anti-HER2 complementary bispecific ADC has a broader spectrum of cell killing activity, not only being able to kill tumor cells that overexpress HER2, but also directly kill tumor cells that underexpress HER2, and also having a killing effect on tumor cells that do not respond to DS-8201.

[0433] To further validate the ability of complementary anti-HER2 bispecific ADCs to kill tumor cells that have become resistant to DS-8201 treatment or have relapsed or refractory disease, this example screened for a tumor cell line (designated SKOV-3R*) with acquired resistance to DS-8201 by continuously subculturing HER2-overexpressing SKOV-3 cells in culture medium containing increasing concentrations of DS-8201. This cell line was able to proliferate normally in culture medium containing 100 nM DS-8201. In vitro cell cytotoxicity assays demonstrated that the complementary bispecific ADCs (ST06-GGFG-Eribulin and ST06-VCP-Eribulin) effectively killed the acquired resistance SKOV-3R* cell line.

[0434] Table 13. In vitro cell killing activity of anti-HER2 complementary bispecific ADC and DS-8201 -: Inactive.

[0435] 5.4 Bystander Killing Effect of Anti-HER2 Complementary Bispecific ADCs

[0436] Anti-HER2 complementary bispecific ADC using HER2-overexpressing cell line BT474 and HER2-non-expressing cell line MDA-MB-468 The bystander killing effect of ST06-GGFG-Eribulin was verified as follows: on day 0, BT474 cells were plated at 10,000 cells / 100 μL / well in a 96-well white-walled plate and cultured overnight at 37°C; on day 1, ST06-GGFG-Eribulin was diluted to 20 nM in culture medium, then serially diluted 5-fold and added to the BT474 cell plate at 100 μL / well (i.e., the initial concentration was 10 nM) and cultured at 37°C; on day 3, MDA-MB-468 cells were plated at 10,000 cells / well in a new 96-well white-walled plate and cultured overnight at 37°C; on day 4, the culture supernatant of the MDA-MB-468 cells was removed, and 100 μL / well of the culture supernatant from the BT474 cell plate (BT474-conditioned medium) was added. Medium) was used as the bystander killing experimental group. Freshly prepared ST06-GGFG-Eribulin, diluted fivefold starting from 10 nM, was added at 100 μL / well as the control group and incubated at 37°C. Simultaneously, BT474 cells were assayed using CellCount Lite 2.0 reagent. After 72 hours of culture, MDA-MB-468 cell viability was assessed using CellCount Lite 2.0 reagent. Data were analyzed using GraphPad Prism 9 software. The results are shown in Figure 20A. After 3 days of treatment with ST06-GGFG-Eribulin, BT474 cells were significantly killed, and MDA-MB-468 cells cultured with its culture supernatant also showed significant cell death. However, the freshly prepared ADC solution had no killing activity against these cells, indicating that while ST06-GGFG-Eribulin kills BT474 cells, the small molecule toxin compound Eribulin released into the culture medium can kill MDA-MB-468 cells that do not express HER2.

[0437] In addition, this example further verified the bystander killing effect of ST06-GGFG-Eribulin by co-culturing BT474 cells and Jurkat cells that do not express HER2. The specific method is as follows: BT474 cells and Jurkat cells expressing GFP were collected and 1×10 5 Cells / well were plated individually or together in a 24-well cell culture plate. ST06-GGFG-Eribulin was then added at a final concentration of 1 nM. After culturing at 37°C for 72 hours, the cells in each well were collected into a fl...

Claims

1. An isolated anti-HER2 antibody or antigen-binding fragment thereof, wherein: The anti-HER2 antibody or antigen-binding fragment thereof has at least one of the following characteristics: (1) The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to HER2 extracellular region subdomains 1, 3 and / or 4; (2) The anti-HER2 antibody or antigen-binding fragment thereof does not affect HER2 and its downstream signal transduction pathways, including not inducing, blocking, or inhibiting HER2-tyrosine residue phosphorylation and / or dephosphorylation, and / or not inducing, blocking, or inhibiting HER2 dimerization and its downstream signal transduction pathways that are dependent on or independent of ligand induction, wherein the HER2 dimerization includes HER2 / HER4 dimerization and HER2 / HER3 dimerization, and the ligands include NRG-1 and Heregulins; as well as (3) The anti-HER2 antibody or antigen-binding fragment thereof does not cross-bind with other members of the human ErbB / HER family (including EGFR, HER3 and HER4).

2. The anti-HER2 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the HER2 extracellular region subdomain 3, and comprises: (1) HCDR1 with an amino acid sequence of X1YGMS (SEQ ID NO: 217), X1=S, N and D; (2) HCDR2 with an amino acid sequence of SISGX2GX3YX4KYX5X6X7VKG (SEQ ID NO: 218), X2=G or S; X3=S or N; X4=T or A; X5=P, A, G or V; X6=D, G, E, P, Q or R; X7=S, K or N; (3) HCDR3 with an amino acid sequence of DYX8GFFDV (SEQ ID NO: 219), X8=A, I, N, R, S or V; (4) an amino acid sequence of RSSQSLX9X 10 SNX 11 LCDR1 of NTYLH (SEQ ID NO: 220), X9 = V or L; X 10 =H or S; X 11 =G, A, I, S, R or T; (5) the amino acid sequence is KVSNRX 12 LCDR2 of S (SEQ ID NO: 221), X 12 =F, D or P; (6) the amino acid sequence is X 13 QSTHVPX 14 LCDR3 of T (SEQ ID NO: 222), X 13 =S or Q; X 14 =Y or W, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

3. The anti-HER2 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising: (1) a HCDR1 as shown in SEQ ID NO: 11, 12, or 13; (2) a HCDR2 as shown in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31; and (3) a HCDR3 as shown in SEQ ID NO: 32, 33, 34, 35, 36, or 37; and (4) a LCDR1 as shown in SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 45; (5) a LCDR2 as shown in SEQ ID NO: 46, 47, or 48; and (6) a LCDR3 as shown in SEQ ID NO: 49, 50, or 51; Furthermore, the anti-HER2 antibody or antigen-binding fragment thereof comprises: (1) HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20, HCDR3 as shown in SEQ ID NO: 32, and LCDR1 as shown in SEQ ID NO: 38, SEQ ID NO: 46 (wherein X 12 =F), LCDR2 shown in SEQ ID NO: 49; or (2) HCDR1 as shown in SEQ ID NO:12 or 13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49; or (3) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:47 or 48, and LCDR3 as shown in SEQ ID NO:49; or (4) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:39, 40, 41, 42, 43, 44, or 45, LCDR2 as shown in SEQ ID NO:46, LCDR3 as shown in SEQ ID NO:49; or (5) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:46, LCDR3 as shown in SEQ ID NO:50 or 51; or (6) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:33, 34, 35, 36, or 37, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:46, LCDR3 as shown in SEQ ID NO:49; or (7) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, LCDR3 as shown in SEQ ID NO:49; or (8) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49; or (9) HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49; or (10) HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 24, 29, 30, or 31, HCDR3 shown in SEQ ID NO:37, LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:

49.

4. The anti-HER2 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the HER2 extracellular region subdomain 1, and comprises: (1) an amino acid sequence of DYX 15 HCDR1, X 15 =S or A; (2) the amino acid sequence is WINTX 16 TGX 17 PTYADX 18 X 19 HCDR2, X 16 =E, N, G, Y or I; X 17 =E, D or S; X 18 =D, K or N; X 19 =F or V; (3) the amino acid sequence is VGX 20 X 21 X 22 HCDR3 of YAMDY (SEQ ID NO: 225), X 20 =R or Y; X 21 =Y or G; X 22 =D or S; (4) the amino acid sequence is X 23 LCDR1 of ASQDVYTAVA (SEQ ID NO:226), X 23 =K or R; (5) the amino acid sequence is X 24 ASX 25 RX 26 LCDR2 of T (SEQ ID NO: 227), X 24 =S, A, D, E, K, L, Q, R, W or Y; X 25 =Y, D, E, K, N, Q, S or T; X 26 =Y, A, E, P or Q; (6) the amino acid sequence is QQX 27 LCDR3 of YSTPPT (SEQ ID NO:228), X 27 =H, S, A or Y, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

5. The anti-HER2 antibody or antigen-binding fragment thereof of claim 1 or 4, comprising: a HCDR1 as shown in SEQ ID NO: 98 or 99; a HCDR2 as shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109; a HCDR3 as shown in SEQ ID NO: 110, 111, 112, or 113; and a LCDR1 as shown in SEQ ID NO: 114 or 115; a LCDR2 as shown in SEQ ID NO: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138; LCDR3 shown in NO:139, 140, 141, or 142; Furthermore, the anti-HER2 antibody or antigen-binding fragment thereof comprises: (1) HCDR1 as set forth in SEQ ID NO:98, HCDR2 as set forth in SEQ ID NO:100, HCDR3 as set forth in SEQ ID NO:110, LCDR1 as set forth in SEQ ID NO:114, LCDR2 as set forth in SEQ ID NO:116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, or 128, LCDR3 as set forth in SEQ ID NO:139; or (2) HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, LCDR3 as shown in SEQ ID NO:140, 141, or 142; or (3) HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, or 109, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, LCDR3 as shown in SEQ ID NO:139; or (4) HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100, HCDR3 as shown in SEQ ID NO:111, 112, or 113, LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, LCDR3 as shown in SEQ ID NO:139; or (5) HCDR1 shown in SEQ ID NO:98, HCDR2 shown in SEQ ID NO:100, HCDR3 shown in SEQ ID NO:110, LCDR1 shown in SEQ ID NO:115, LCDR2 shown in SEQ ID NO:116, LCDR3 shown in SEQ ID NO:139; or (6) HCDR1 shown in SEQ ID NO:99, HCDR2 shown in SEQ ID NO:100, HCDR3 shown in SEQ ID NO:110, LCDR1 shown in SEQ ID NO:114, LCDR2 shown in SEQ ID NO:116, LCDR3 shown in SEQ ID NO:139; or (7) a HCDR1 as set forth in SEQ ID NO:98, a HCDR2 as set forth in SEQ ID NO:102, a HCDR3 as set forth in SEQ ID NO:110, a LCDR1 as set forth in SEQ ID NO:115, a LCDR2 amino acid sequence as set forth in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, or a LCDR3 as set forth in SEQ ID NO:142; Preferably, the anti-HER2 antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:129, 133, 136, or 138, and LCDR3 as shown in SEQ ID NO:

142.

6. The anti-HER2 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to HER2 extracellular region subdomain 4, which comprises: HCDR1 as shown in SEQ ID NO: 199, HCDR2 as shown in SEQ ID NO: 200, HCDR3 as shown in SEQ ID NO: 201, and LCDR1 as shown in SEQ ID NO: 202, LCDR2 as shown in SEQ ID NO: 203 and LCDR3 as shown in SEQ ID NO: 204; or, The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the HER2 extracellular region subdomain 1, which comprises: HCDR1 as shown in SEQ ID NO: 205, HCDR2 as shown in SEQ ID NO: 206, HCDR3 as shown in SEQ ID NO: 207, and LCDR1 as shown in SEQ ID NO: 208, LCDR2 as shown in SEQ ID NO: 209 and LCDR3 as shown in SEQ ID NO: 210; or; The anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the HER2 extracellular region subdomain 1, which comprises: HCDR1 as shown in SEQ ID NO: 211, HCDR2 as shown in SEQ ID NO: 212, HCDR3 as shown in SEQ ID NO: 213, and HCDR4 as shown in SEQ ID NO:

214. LCDR1 shown in NO:214, LCDR2 shown in SEQ ID NO:215 and LCDR3 shown in SEQ ID NO:

216.

7. The anti-HER2 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising: a heavy chain variable region VH having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, and a light chain variable region VL having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97; Furthermore, the anti-HER2 antibody or antigen-binding fragment thereof comprises: (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 1 and the VL set forth in SEQ ID NO: 2, respectively; or (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:52 and the VL set forth in SEQ ID NO:81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, respectively; or (3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 and the VL set forth in SEQ ID NO:81, respectively; or (4) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 and the VL set forth in SEQ ID NO: 92, respectively; Preferably, the anti-HER2 antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 73, 78, 79, or 80 and the VL shown in SEQ ID NO: 92, respectively.

8. The anti-HER2 antibody or antigen-binding fragment thereof of claim 1, 4 or 5, comprising: a heavy chain variable region VH having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 3, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170, and a heavy chain variable region VH having at least 80% identity to the amino acid sequence shown in SEQ ID NO: NO:4, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 having at least 80% identity to a light chain variable region VL; Furthermore, the anti-HER2 antibody or antigen-binding fragment thereof comprises: (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:3 and the VL set forth in SEQ ID NO:4, respectively; or (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 143 and the VL set forth in SEQ ID NO: 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188, respectively; or (3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 170 and the VL set forth in SEQ ID NO: 171, respectively; or (4) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO: 169 and the VL set forth in SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively; Preferably, the anti-HER2 antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively; More preferably, the anti-HER2 antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NO: 189, 193, 196, or 198, respectively.

9. The anti-HER2 antibody or antigen-binding fragment thereof according to claim 1 or 6, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH and the light chain variable region VL comprise: (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:5 and the VL set forth in SEQ ID NO:6, respectively; or, (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:7 and the VL set forth in SEQ ID NO:8, respectively; or, (3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH set forth in SEQ ID NO:9 and the VL set forth in SEQ ID NO:10, respectively.

10. An anti-HER2 complementary bispecific antibody, comprising two antigen binding domains capable of simultaneously and non-competitively binding to the extracellular region of HER2, wherein the first antigen binding domain and the second antigen binding domain each specifically bind to a different epitope in the extracellular region of HER2, and the epitope is located in subdomains 1, 3 and 4 of the extracellular region of HER2, and the complementary bispecific antibody comprises at least one of the following characteristics: (1) The complementary bispecific antibody can cross-link / aggregate HER2 on the surface of tumor cells, thereby inducing rapid and efficient internalization of the HER2 cross-linker, as shown by an internalization rate of more than 60% in HER2 low-expressing tumor cells and more than 80% in HER2 over-expressing tumor cells; (2) The complementary bispecific antibody can effectively trigger the lysosomal transport and degradation of HER2 on the surface of tumor cells, significantly reduce the expression of HER2 on the cell surface, and thus significantly inhibit the proliferation of HER2-overexpressing tumor cells; (3) The complementary bispecific antibody does not affect HER2 and its downstream signal transduction pathways, including not inducing, blocking, or inhibiting ligand-induced HER2 dimerization and activation of its downstream signal transduction pathways. Therefore, the complementary bispecific antibody does not affect the function and regulation of HER2 and its downstream signal transduction pathways in normal tissues / cells, including cardiomyocytes. The ligand includes NRG-1. (4) The complementary bispecific antibody can specifically bind to HER2 without cross-binding to other members of the human ErbB / HER family (including EGFR, HER3 and HER4).

11. The complementary bispecific antibody according to claim 10, wherein: One of the first antigen-binding domain and the second antigen-binding domain specifically binds to the HER2 extracellular region subdomain 1, and the other specifically binds to the HER2 extracellular region subdomain 3; Furthermore, the first antigen-binding domain comprises: (1) HCDR1 with an amino acid sequence of X1YGMS, where X1=S, N and D; (2) HCDR2 with an amino acid sequence of SISGX2GX3YX4KYX5X6X7VKG, where X2=G or S; X3=S or N; X4=T or A; X5=P, A, G or V; X6=D, G, E, P, Q or R; X7=S, K or N; (3) HCDR3 with an amino acid sequence of DYX8GFFDV, where X8=A, I, N, R, S or V; (4) an amino acid sequence of RSSQSLX9X 10 SNX 11 LCDR1 of NTYLH, X9 = V or L; X 10 =H or S; X 11 =G, A, I, S, R or T; (5) the amino acid sequence is KVSNRX 12 LCDR2 of S, X 12 =F, D or P; (6) the amino acid sequence is X 13 QSTHVPX 14 LCDR3 of T, X 13 =S or Q; X 14 =Y or W, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; The second antigen-binding domain comprises: (1) an amino acid sequence of DYX 15 MH HCDR1, X 15 =S or A; (2) the amino acid sequence is WINTX 16 TGX 17 PTYADX 18 X 19 HCDR2 of KG, X 16 =E, N, G, Y or I; X 17 =E, D or S; X 18 =D, K or N; X 19 =F or V; (3) the amino acid sequence is VGX 20 X 21 X 22 YAMDY's HCDR3, X 20 =R or Y; X 21 =Y or G; X 22 =D or S; (4) the amino acid sequence is X 23 ASQDVYTAVA LCDR1,X 23 =K or R; (5) the amino acid sequence is X 24 ASX 25 RX 26 LCDR2 of T, X 24 =S, A, D, E, K, L, Q, R, W or Y; X 25 =Y, D, E, K, N, Q, S or T; X 26 =Y, A, E, P or Q; (6) the amino acid sequence is QQX 27 LCDR3 of YSTPPT, X 27 =H, S, A or Y, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

12. The anti-HER2 complementary bispecific antibody according to claim 10 or 11, wherein: The first antigen binding domain comprises: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49; The second antigen binding domain comprises: a HCDR1 as shown in SEQ ID NO:98, a HCDR2 as shown in SEQ ID NO:102, a HCDR3 as shown in SEQ ID NO:110, and a LCDR1 as shown in SEQ ID NO:115, a LCDR2 as shown in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and a LCDR3 as shown in SEQ ID NO:142; Further, the first antigen binding domain comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:24, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49; The second antigen binding domain comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133 and LCDR3 as shown in SEQ ID NO:

142.

13. The anti-HER2 complementary bispecific antibody according to claim 10 or 11, wherein: The first antigen-binding domain comprises: a VH as shown in SEQ ID NO: 70, 72, 73, 76, 77, 78, 79, or 80, and a VL as shown in SEQ ID NO: 92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively; the second antigen-binding domain comprises: a VH as shown in SEQ ID NO: 169, and a VL as shown in SEQ ID NO:

92. NO:189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively; Preferably, the first antigen-binding domain comprises: a VH as shown in SEQ ID NO:73, 78, 79, or 80, and a VL as shown in SEQ ID NO:92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively; and the second antigen-binding domain comprises: a VH as shown in SEQ ID NO:169, and a VL as shown in SEQ ID NO:193, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said VH and VL, respectively.

14. The anti-HER2 complementary bispecific antibody according to any one of claims 10 to 12, wherein the structure of the bispecific antibody is in the form of DVD-Ig, wherein: One of the first antigen-binding domain or the second antigen-binding domain is an Fv domain, and the other is a Fab or IgG domain, and the Fv domain is connected to the Fab or IgG domain via a linker; Further, the first antigen binding domain specifically binds to the HER2 extracellular region subdomain 3 and is an Fv domain, the second antigen binding domain specifically binds to the HER2 extracellular region subdomain 1 and is a Fab domain or an IgG domain, the first or second antigen binding domain comprises a heavy chain variable region VH and a light chain variable region VL, the C-terminus of the VH domain in the Fv domain is connected to the N-terminus of the VH domain in the Fab or IgG domain through a linker, and the C-terminus of the VL domain in the Fv domain is connected to the N-terminus of the VL domain in the Fab or IgG domain through a linker.

15. The anti-HER2 complementary bispecific antibody according to any one of claims 10 to 14, comprising 4 polypeptide chains: (1) wherein the two polypeptide chains comprise VH1-L1-VH2-C-(Fc)n, wherein VH1 represents a heavy chain variable region that specifically binds to a first antigen binding domain of the HER2 extracellular region subdomain 3, L1 represents a linker, VH2 represents a heavy chain variable region that specifically binds to a second antigen binding domain of the HER2 extracellular region subdomain 1, C represents a heavy chain constant region CH1, Fc represents a heavy chain constant region Fc domain, and n is 0 or 1; and (2) the other two polypeptide chains comprise VL1-L2-VL2-CL, wherein VL1 represents a light chain variable region of a first antigen binding domain that specifically binds to subdomain 3 of the extracellular region of HER2, L2 represents a linker, VL2 represents a light chain variable region of a second antigen binding domain that specifically binds to subdomain 1 of the extracellular region of HER2, and CL is an IgG light chain constant region; The L1 and L2 linkers comprise (G4S) n Sequence, n represents the copy number of G4S, which is an integer greater than 0, and the copy numbers n of L1 and L2 linkers can be the same or different.

16. The anti-HER2 complementary bispecific antibody according to claim 15, wherein: The VH1 comprises the HCDR1 shown in SEQ ID NO:11, the HCDR2 shown in SEQ ID NO:24, 29, 30, or 31, and the HCDR3 shown in SEQ ID NO:37; VH2 comprises the HCDR1 shown in SEQ ID NO:98, the HCDR2 shown in SEQ ID NO:102, and the HCDR3 shown in SEQ ID NO:110; the VL1 comprises the LCDR1 shown in SEQ ID NO:42, the LCDR2 shown in SEQ ID NO:46, and the LCDR3 shown in SEQ ID NO:49; VL2 comprises the LCDR1 shown in SEQ ID NO:115, the LCDR2 shown in SEQ ID NO:133, and the LCDR3 shown in SEQ ID NO:

142.

17. The anti-HER2 complementary bispecific antibody according to claim 15 or 16, wherein: The VH1 comprises a VH as set forth in SEQ ID NO:73, 78, 79, or 80, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the VH2 comprises a VH as set forth in SEQ ID NO:169, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; the VL1 comprises a VH as set forth in SEQ ID NO:169, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; NO:92, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the VL2 comprises the VL as shown in SEQ ID NO:193, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

18. The anti-HER2 complementary bispecific antibody according to any one of claims 15 to 17, wherein: The L1 comprises the linker amino acid sequence shown in SEQ ID NO: 229; the L2 comprises the linker amino acid sequence shown in SEQ ID NO: 230; the Fc comprises an engineered Fc domain, preferably, comprising the amino acid sequence of human IgG1 Fc with L234F / L235E / P331S (EU numbering system) amino acid substitutions as shown in SEQ ID NO: 232; the CL is selected from a human κ constant region or a human λ constant region, preferably, the human κ constant region amino acid sequence as shown in SEQ ID NO:

233.

19. An anti-HER2 complementary bispecific antibody-drug conjugate (ADC), comprising an anti-HER2 complementary bispecific antibody according to any one of claims 10 to 18, a small molecule toxin compound, and a cleavable linker, wherein the ADC comprises at least one of the following properties: (1) The ADC can simultaneously bind to two different epitopes of HER2 on the surface of tumor cells, cross-link HER2 to form clusters, and trigger rapid endocytosis and lysosomal transport of the ADC, thereby significantly improving the entry of small molecule toxin compounds in the ADC into target cells and their release in target cells. The efficiency of the ADC is such that the ADC has a broader spectrum of killing activity, which is manifested in that it not only has a stronger killing activity than existing HER2 targeted drugs in tumor cells with HER2 overexpression, but also can play a direct killing role in tumor cells with low HER2 expression. The existing HER2 targeted drugs include Trastuzumab, Pertuzumab, T-DM1 and DS-8201. (2) The ADC has a killing effect on tumors that have become resistant or recurrent to the above-mentioned existing HER2 targeted drugs; (3) The ADC does not affect the biological activity of HER2 and the regulation of its mediated signal transduction pathway, does not interfere with the normal function of HER2 in cardiomyocytes, and does not have Fc effector function. Therefore, its clinical safety is significantly higher than the above-mentioned existing HER2 targeted drugs; Furthermore, the ADC is represented by formula (I): Ab-(LD) p (Ⅰ) in, Ab represents an anti-HER2 complementary bispecific antibody as described in any one of claims 10 to 18, wherein the Ab comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to the HER2 extracellular region subdomain 3 and is an Fv domain, and the second antigen-binding domain specifically binds to the HER2 extracellular region subdomain 1 and is an IgG domain, and the first or second antigen-binding domain comprises a heavy chain variable region and a light chain variable region; D represents a small molecule toxin compound; L represents a cleavable linker; and p is 2 to 8.

20. The ADC of claim 19, wherein: The first antigen-binding domain comprises: (1) HCDR1 as shown in SEQ ID NO:11, (2) HCDR2 as shown in SEQ ID NO:24, 29, 30, or 31, (3) HCDR3 as shown in SEQ ID NO:37, (4) LCDR1 as shown in SEQ ID NO:42, (5) LCDR2 as shown in SEQ ID NO:46, and (6) LCDR3 as shown in SEQ ID NO:49; the second antigen-binding domain comprises: (1) HCDR1 as shown in SEQ ID NO:98, (2) HCDR2 as shown in SEQ ID NO:102, (3) HCDR3 as shown in SEQ ID NO:110, (4) LCDR1 as shown in SEQ ID NO:115, (5) LCDR2 as shown in SEQ ID NO:133, and (6) LCDR3 as shown in SEQ ID NO:

142.

21. The ADC of claim 19 or 20, wherein: The first antigen-binding domain comprises a VH as shown in SEQ ID NO:73, 78, 79, or 80 and a VL as shown in SEQ ID NO:92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively; the second antigen-binding domain comprises a VH as shown in SEQ ID NO:169 and a VL as shown in SEQ ID NO:193, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.

22. The ADC of any one of claims 19-21, wherein: The Ab further comprises a constant region, which includes a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises human IgG1 Fc as shown in SEQ ID NO: 232, and the light chain constant region comprises a human κ constant region as shown in SEQ ID NO:

233.

23. The ADC of any one of claims 19-22, wherein: The small molecule toxin compounds include Eribulin, Auristatin derivatives, Tubulysins, Cryptomycins, Maytansine derivatives, topoisomerase inhibitors, Pyrrolobenzodiazepines (PBDs), Calicimycin and its derivatives, Duocarmycins, alkyl chemotherapeutic agents and other compounds with alkylated forms, antimetabolites, mitotic inhibitors, and cytotoxic antibiotics; Furthermore, the auristatin derivatives include MMAE, MMAF, and MMAD; the maytansine derivatives include DM1, DM2, DM3, and DM4; the topoisomerase inhibitors include camptothecin derivatives SN-38, exatecan, and DXd; the alkyl chemotherapeutic agents and other compounds with alkylated forms include nitrogen mustards, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, and dacarbazine; the antimetabolites include folic acid, purine or pyrimidine antagonists; the mitotic inhibitors include derivatives of vinca alkaloids and podophyllotoxin; the cytotoxic antibiotics include anthracycline antibiotics, actinomycin, and bleomycin; and the preferred small molecule toxin compound is eribulin.

24. The ADC of any one of claims 19-23, wherein: The cleavable linker comprises a cleavable peptide portion and at least one spacer; Further, the cleavable peptide portion comprises an amino acid unit comprising a Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG) amino acid sequence cleaved by cathepsin B; The spacer comprises a spacer conjugated to an antibody, and / or a second spacer conjugated to a small molecule toxin compound, wherein the spacer conjugated to the antibody is a hydrophilic spacer comprising one or more polyethylene glycols (PEG), and the second spacer comprises a p-aminobenzyl unit.

25. The ADC of claim 24, wherein the amino acid unit comprises VC and GGFG; The hydrophilic spacer comprises two PEGs, and the hydrophilic spacer and maleimide (Mal) form a Mal-spacer to be attached to the antibody; the second spacer comprises p-aminobenzylcarbonyl (PAB).

26. The ADC of claim 24 or 25, wherein the cleavable linker comprises a Mal-spacer and a cleavable peptide portion, the Mal-spacer comprises 2 PEGs, the cleavable peptide portion comprises a dipeptide VC and a tetrapeptide GGFG; or the cleavable linker comprises a Mal-spacer, a cleavable peptide portion and a second spacer, the Mal-spacer comprises 2 PEGs, the cleavable peptide portion comprises a dipeptide VC, and the second spacer comprises PAB.

27. The ADC of any one of claims 24-26, wherein the cleavable linker comprises Mal-(PEG)2-VC, Mal-(PEG)2-GGFG, or Mal-(PEG)2-VC-PAB.

28. The ADC of any one of claims 24-27, wherein the cleavable peptide portion of the cleavable linker can be coupled to the small molecule toxin compound directly or through a second spacer to prepare an ADC containing a drug payload, wherein the small molecule toxin compound is eribulin.

29. The ADC of claim 28, wherein the cleavable linker comprises Mal-(PEG)2-GGFG, and the drug payload comprises the compound Mal-(PEG)2-GGFG-Eribulin obtained by coupling the cleavable peptide portion GGFG of the cleavable linker to the C-35 amine of Eribulin via a carboxyl group; or The cleavable linker comprises Mal-(PEG)2-VC-PAB, and the drug payload comprises the compound Mal-(PEG)2-VC-PAB-Eribulin obtained by coupling the cleavable linker to the C-35 amine of Eribulin via a second spacer region PAB.

30. The ADC of any one of claims 19-29, wherein p is 4 to 8.

31. A nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the anti-HER2 complementary bispecific antibody according to any one of claims 10 to 18, or the antibody portion of the ADC according to any one of claims 19 to 30.

32. An expression vector capable of expressing the nucleic acid of claim 31.

33. A host cell comprising the nucleic acid of claim 31 or the expression vector of claim 32.

34. A method for preparing the anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the anti-HER2 complementary bispecific antibody according to any one of claims 10 to 18, or the antibody portion of the ADC according to any one of claims 19 to 30 using the host cell according to claim 33, comprising: (i) expressing the anti-HER2 antibody or its antigen-binding fragment, the anti-HER2 complementary bispecific antibody, or the antibody portion of the ADC in the host cell, and (ii) isolating the anti-HER2 antibody or its antigen-binding fragment, the anti-HER2 complementary bispecific antibody, or the antibody portion of the ADC from the host cell or cell culture.

35. A method for preparing the ADC according to any one of claims 19 to 30, comprising: The nucleophilic group or electrophilic group of the small molecule toxin compound reacts with the cleavable linker to form a drug payload via a covalent bond, and then reacts with the nucleophilic group or electrophilic group of the antibody, or preferably, the free cysteine ​​residue generated after the interchain disulfide bond of the antibody hinge region is reduced and reacts with the reactive functional group of the drug payload to form a covalent bond, and the reactive functional group includes a maleimide group.

36. A pharmaceutical composition comprising the anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the anti-HER2 complementary bispecific antibody according to any one of claims 10 to 18, or the ADC according to any one of claims 19 to 30, and a pharmaceutically acceptable carrier.

37. A drug kit comprising an effective amount of the anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the anti-HER2 complementary bispecific antibody according to any one of claims 10 to 18, the anti-HER2 complementary bispecific ADC according to any one of claims 19 to 30, or the pharmaceutical composition according to claim 36, and optionally at least one additional tumor therapeutic agent; Further, the additional tumor therapeutic agents include HER2 antagonists, EGFR antagonists, HER3 antagonists, MET antagonists, IGF1R antagonists, B-Raf inhibitors, PDGFR-α inhibitors, PDGFR-β inhibitors, PDGF ligand inhibitors, VEGF antagonists, VEGF receptor kinase inhibitors, DLL4 antagonists, Ang2 antagonists, FOLH1 antagonists, STEAP1 or STEAP2 antagonists, TMPRSS2 antagonists, MSLN antagonists, MUC16 antagonists, CLEC12A antagonists, PD-1 or PD-L1 blockers, hormone receptor modulators, aromatase inhibitors, kinase inhibitors, cytokine agonists or cytokine inhibitors, and chemotherapeutic agents.

38. A method for treating a cancer expressing HER2, comprising administering to a subject in need thereof an effective amount of the anti-HER2 complementary bispecific antibody of any one of claims 10-18, the anti-HER2 complementary bispecific ADC of any one of claims 19-30, the pharmaceutical composition of claim 36, or the kit of claim 37.

39. The method of claim 38, wherein: The cancers include breast cancer, ovarian cancer, cervical cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, melanoma, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer and endometrial cancer, and the cancers also include cancers at various stages, such as early cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer or cancer in remission; the cancers also include cancers with overexpression of HER2, cancers with relatively low expression levels of HER2, cancers that do not respond or respond poorly to existing HER2 targeted therapeutics, and / or cancers that are resistant to or relapsed from existing HER2 targeted therapeutics, wherein the existing HER2 targeted therapeutics include Trastuzumab, Pertuzumab, T-DM1 and DS-8201; The subject may be a human, non-human primate or other mammal such as a dog, mouse, or rat; the subject further includes patients who are not suitable for or are difficult to treat with existing HER2 targeted therapies, or who develop drug resistance or relapse after receiving existing HER2 targeted therapies.

40. A method for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, and a method for screening cancer patients responsive to ADC treatment as described in any one of claims 19-30, comprising incubating the anti-HER2 antibody or antigen-binding fragment thereof as described in any one of claims 1-9, or the anti-HER2 complementary bispecific antibody as described in any one of claims 10-18 with the sample or a biological sample isolated from the patient, and detecting whether the antibody binds to the sample or biological sample.

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