Anti-HER2 biparatopic antibody-drug conjugate, method for preparing the same, and its use

Abstract

Provided are anti-HER2 antibodies or their antigen-binding fragments, or anti-HER2 biparatopic (complementary bispecific) antibodies and biparatopic antibody-drug conjugates (ADCs) constructed from them, as well as pharmaceutical compositions and kits containing them. Methods for using such antibodies, ADCs, pharmaceutical compositions and kits to treat HER2 abnormality-related diseases are also provided.

Description

[Technical Field]

[0001] The present invention relates to anti-HER2 antibodies or antigen-binding fragments thereof, as well as anti-HER2 biparatopic (complementary bispecific) antibodies derived therefrom, and any antibody-drug conjugates containing such antibodies. The present invention also provides methods for preparing biparatopic antibodies and their antibody-drug conjugates, and methods for their use in the treatment of diseases associated with abnormal HER2 expression. [Background technology]

[0002] Human epidermal growth factor receptor 2 (HER2 or ErbB2) is a member of the ErbB / HER family of receptor tyrosine kinases. Receptors of the ErbB / HER family activate downstream signaling pathways after dimerization, thereby participating in the regulation of cell proliferation, differentiation, and survival. HER2 is a transmembrane glycoprotein with a molecular weight of approximately 185 kDa, consisting of an extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Mutations and / or amplification of the ErbB2 / HER2 gene can lead to HER2 overexpression, which can be detected by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays. + or IHC2 + / FISH + It is classified as such. Clinical trials have shown that HER2 is overexpressed in various cancers (e.g., breast cancer, ovarian cancer, endometrial cancer, gastric cancer, prostate cancer, and lung cancer), and that HER2 overexpression is associated with tumor aggressiveness and poor prognosis.

[0003] Several HER2-targeted therapies have been approved to treat HER2-overexpressing breast and gastric cancers, and these have significantly improved patient survival rates. However, a considerable number of cancer patients do not respond to these therapies. Furthermore, existing HER2-targeted therapies have the following drawbacks, which limit their clinical efficacy and / or pose safety risks.

[0004] (1) Current HER2-targeted therapies are IHC2 + / FISH - or IHC1 + This approach is ineffective in eradicating cancer cells that express relatively low levels of HER2, such as those classified as "low-HER2 expression." Tumor cells expressing low levels of HER2 often acquire resistance to existing treatments due to the greater heterogeneity of HER2 expression compared to HER2-overexpressing tumors. As a result, most cancer patients develop drug resistance and experience disease progression 6 to 12 months after receiving current HER2-targeted therapy.

[0005] (2) Approximately 70% of breast cancer patients are classified as HER2-low expression. Currently, DS-8201 is the only HER2-targeted drug approved for this patient population, but in the Phase III DESTINY-Breast04 clinical trial, it showed only a response rate of about 50%, and the incidence of drug-related interstitial lung disease / pneumonia was 12.1% (Modi et al., N Engl J Med 2022, 387:9-20). Clearly, there is a considerable urgent need to improve the efficacy and safety of current HER2-targeted therapies for treating HER2-low expression tumors.

[0006] (3) The HER2-mediated signaling pathway is important for maintaining cardiomyocyte survival, repairing cardiomyocyte damage, and maintaining cardiomyocyte functional integrity. Currently approved anti-HER2 therapies, including antibodies, ADCs, and small molecule inhibitors, can disrupt HER2 signaling and lead to cardiotoxicity. For example, trastuzumab and pertuzumab can cause a decrease in left ventricular ejection fraction (LVEF) and congestive heart failure. Trastuzumab, in particular, carries a higher risk of inducing heart failure when administered concomitantly with anthracyclines, and therefore requires rigorous screening of eligible patients or prophylactic cardioprotective therapy before treatment with these drugs.

[0007] Therefore, in order to overcome the safety and resistance limitations of current treatments, there remains a critical unmet clinical need to develop safer (particularly addressing the cardiotoxicity caused by current HER2-targeted therapies) and more potent targeted therapies for HER2-low-expressing cancer cells. [Overview of the Initiative] [Means for solving the problem]

[0008] This invention provides a class of HER2-targeted biparatopic antibody-drug conjugates. In one embodiment, a biparatopic ADC can specifically bind to two different epitopes on the extracellular domain (also referred to as the "extracellular region") of HER2, which cross-links HER2 on the cell surface to form a reticular multimer, thereby promoting rapid and efficient endocytosis and altering the intracellular endosomal transport pathway from recycling to lysosomal transport. As a result, the ADC bound to the cell surface is efficiently (almost 100%) transported to lysosomes and degraded, thus releasing more small molecule toxins into the cytoplasm to exert cytotoxic and subsequent bystander-killing effects. In this way, the biparatopic ADC exhibits a broader spectrum of target cell-killing activity. Not only can it kill HER2-overexpressing cancer cells, but it can also directly kill cancer cells expressing low levels of HER2 (IHC2+ / FISH- or IHC1+), thereby significantly reducing the probability of acquiring drug resistance due to heterogeneity of HER2 expression in tumors. In another aspect, the binding epitopes of biparatopic ADCs, unlike those of trastuzumab and pertuzumab, do not interfere with HER2 dimerization or affect the modulation of HER2 downstream signaling pathways; therefore, biparatopic ADCs do not interfere with the normal biological function of HER2 on cardiomyocytes.

[0009] In one embodiment, the present invention provides high binding activity or binding affinity (e.g., binding affinity constant [K]) to HER2-expressing tumor cells. D The value of ] is <1 × 10 -8M, <5×10 -9 M, or <1×10 -9 M), and has no cross-reactivity with other members of the ErbB / HER family (including EGFR, HER3, and HER4), and provides an isolated anti-HER2 antibody or antigen-binding fragment thereof that can specifically bind to the extracellular domain of HER2. The anti-HER2 antibody or antigen-binding fragment thereof has no antagonist activity or agonist activity against HER2 or its downstream signaling pathway.

[0010] In some embodiments, the anti-HER2 antibody includes anti-mouse HER2 antibodies and chimeric antibodies derived therefrom, humanized antibodies, and / or optimized antibodies. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof specifically binds to subdomain I, III, or IV of the HER2 extracellular domain, preferably subdomain I or III. In some embodiments, the paired anti-HER2 antibodies or antigen-binding fragments thereof used to construct the anti-HER2 bivalent antibody specifically recognize multiple different epitopes on the extracellular domain of HER2 and do not compete with each other for binding.

[0011] In one aspect, the present invention provides an anti-HER2 bivalent antibody comprising a first antigen-binding domain and a second antigen-binding domain, each antigen-binding domain specifically recognizes a different epitope on HER2, and enables the bivalent antibody to cross-link HER2 on the cell surface and induce cluster formation. The formation of clusters is highly dependent on the HER2 density on the cell surface. The formation of clusters on the cell surface will result in rapid internalization and lysosomal degradation. Compared with the monospecific anti-HER2 antibodies and / or trastuzumab described in the present invention, the bivalent antibody can bring about significantly enhanced HER2 internalization and lysosomal transport / degradation.

[0012] In some embodiments, the first and second antigen-binding domains of an anti-HER2 biparatopic antibody specifically bind to two distinct (non-overlapping or non-competitive) epitopes on HER2, the epitopes comprising sequences located in subdomains I, III, and / or IV of the HER2 extracellular domain. In one embodiment, the first antigen-binding domain specifically binds to subdomain III of the HER2 extracellular domain, and the second antigen-binding domain specifically binds to subdomain I of the HER2 extracellular domain, and both epitopes are distinct from the epitopes bound by trastuzumab or pertuzumab. In one embodiment, the biparatopic antibody neither inhibits nor activates HER2 or its downstream signaling pathways within the cell. In one embodiment, the biparatopic antibody, upon binding to and crosslinking HER2 on the cell surface, induces rapid receptor internalization and efficient lysosomal transport. In one embodiment, the biparatopic antibody can induce degradation of HER2 on the cell surface. In one embodiment, a biparatopic antibody induces HER2 internalization and lysosomal degradation by crosslinking HER2 on the cell surface, significantly reducing HER2 expression on the cell surface and thus effectively inhibiting the proliferation of HER2-overexpressing tumor cells.

[0013] In one embodiment, the present invention provides an ADC comprising a small molecule toxin compound conjugated via a linker to a disclosed anti-HER2 biparatopic antibody. The ADC can specifically bind to and crosslink HER2 on the surface of tumor cells, thereby forming aggregated "clusters," leading to rapid internalization and lysosomal transport, as well as ADC degradation in lysosomes, resulting in a significant increase in the release of toxin into the cytoplasm of tumor cells. Thus, the biparatopic ADC exhibits a broader spectrum of tumor cell-killing activity compared to T-DM1 and / or DS-8201, i.e., the biparatopic ADC can effectively kill both HER2-overexpressing and HER2-lowexpressing tumor cells. The ADC has the following formula (I): Ab-(LD)p (I) During the ceremony, Ab is the anti-HER2 biparatopic antibody of the present invention, D is a small molecule toxin compound (drug), L is a cleavable linker that connects Ab to D, p represents the copy number of the (LD) bonded to Ab, and is in the range of 2 to 8.

[0014] In some embodiments, the small molecule toxin compound comprises a cytotoxin and a chemotherapeutic agent. In one embodiment, the small molecule toxin compound is a cytotoxin comprising a tubulin inhibitor and a DNA alkylating agent. Preferably, the tubulin inhibitor includes eribulin, auristatin derivatives (e.g., MMAE, MMAF, MMAD), tubulisin, cryptomycin, and maytansinoid derivatives (e.g., DM1, DM2, DM3, DM4), and the DNA alkylating agent includes topoisomerase inhibitors (e.g., camptothecin derivatives such as SN-38, exatecan, and DXd [exatecan derivative for ADCs]), pyrrolobenzodiazepine (PBD), calicheamycin and its derivatives (e.g., N-acetylcalicheamycin [CMC]), and duocalmycin. In one embodiment, the small molecule toxin compound is eribulin.

[0015] In some embodiments, the cleavable linker may be any linker containing a cleavable moiety, the cleavable moiety containing any cleavable chemical bond. In some specific embodiments, the cleavable linker contains a cleavable peptide moiety that can be cleaved by an intracellular peptidase or protease, the cleavable peptide moiety containing amino acid units such as a dipeptide, tripeptide, or tetrapeptide. In some specific embodiments, the cleavable linker contains at least one spacer that binds the anti-HER2 biparatopic antibody (Ab) of the present invention to a small molecule toxin compound (D), the spacer containing one spacer bound to the antibody and / or a second spacer bound to the small molecule toxin compound. In some embodiments, the spacer bound to the antibody is hydrophilic, and exemplary spacers contain polyethylene glycol (PEG). In some examples, the spacer is bound to the anti-HER2 biparatopic antibody of the present invention via a maleimide moiety (Mal). In some embodiments, the second spacer helps to link the cleavable moiety (e.g., a cleavable peptide) of the cleavable linker to the small molecule toxin compound. In some examples, the second spacer bound to the small molecule toxin compound is self-degradable, and the self-degradable spacer contains a p-aminobenzyl unit. In some specific embodiments, the cleavable linker includes a Mal-spacer and a cleavable peptide moiety. In some specific embodiments, the Mal-spacer in the cleavable linker is bound to one or more amino acid residues of the antibody component of the ADC of the present invention. In some embodiments, the maleimide group of the Mal-spacer can react with the thiol group of a cysteine ​​residue at a specific position in the constant and / or variable regions of the antibody. In some specific embodiments, the second spacer in the cleavable linker is bound to the small molecule toxin compound of the ADC of the present invention, and the small molecule toxin compound is eribulin or a derivative thereof. In some specific embodiments, the cleavable moiety (e.g., a cleavable peptide) in the cleavable linker is directly bound to the small molecule toxin compound of the ADC, and the small molecule toxin compound is eribulin or a derivative thereof.

[0016] In some embodiments, p is in the range of 2 to 8, for example, 4 to 8. As used herein, p is an integer greater than 0 or a non-integer.

[0017] In another aspect, the present invention relates to an anti-HER2 biparatopic antibody and an isolated nucleic acid molecule (also called a "polynucleotide") encoding the corresponding monospecific antibody or antigen-binding fragment thereof, as well as an expression vector containing said nucleic acid molecule, and a host cell containing said nucleic acid molecule or expression vector. The present invention also relates to a method for preparing the anti-HER2 monospecific antibody or antigen-binding fragment and the anti-HER2 biparatopic antibody disclosed herein using said host cell, the method comprising culturing said host cell and recovering said antibody or antigen-binding fragment from the culture medium.

[0018] In another embodiment, the present invention relates to a pharmaceutical composition comprising an anti-HER2 monospecific antibody or its antigen-binding fragment, an anti-HER2 biparatopic antibody, or an anti-HER2 biparatopic antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier.

[0019] In another embodiment, the present invention relates to a kit comprising an effective amount of the anti-HER2 monospecific antibody or its antigen-binding fragment, anti-HER2 biparatopic antibody, anti-HER2 biparatopic antibody-drug conjugate or pharmaceutical composition, and optionally at least one additional anticancer agent.

[0020] In another embodiment, the present invention relates to a method for treating a target HER2-expressing tumor, comprising administering the anti-HER2 biparatopic antibody, anti-HER2 biparatopic antibody-drug conjugate, pharmaceutical composition, or kit of the present invention to a target subject as needed. Alternatively, the present invention relates to the use of the anti-HER2 biparatopic antibody, anti-HER2 biparatopic antibody-drug conjugate, pharmaceutical composition, or kit described herein in the manufacture of a pharmacopoeia for treating a HER2-expressing tumor in a subject. Alternatively, the present invention relates to the use of the anti-HER2 biparatopic antibody, anti-HER2 biparatopic antibody-drug conjugate, pharmaceutical composition, or kit described herein in the treatment of a HER2-expressing tumor in a subject. In some embodiments, the tumor is a HER2-overexpressing tumor (IHC3). + or IHC2 + / FISH + ) and / or low HER2-expressing tumors (IHC2 + / FISH - , or IHC1 + ) include. In some embodiments, the anti-HER2 biparatopic antibody-drug conjugate or its pharmaceutical composition exhibits cytotoxic effects against HER2-overexpressing and / or low-HER2-expressing tumor cells. In some embodiments, the subjects are mammals. 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 are HER2-overexpressing or low-HER2-expressing.

[0021] In some embodiments, the tumor is resistant to existing HER2-targeted therapies. In some embodiments, the tumor is resistant to HER2-targeted therapeutics including trastuzumab, pertuzumab, T-DM1, and / or DS-8201. In one embodiment, the tumor is unresponsive or poorly responsive to HER2-targeted therapeutics including trastuzumab, pertuzumab, T-DM1, DS-8201, and / or taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, etc.).

[0022] In some embodiments, the subjects include patients who are ineligible for or refractory to existing HER2-targeted therapies, or who have developed resistance to or relapsed to existing HER2-targeted therapies.

[0023] 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 may respond to treatment with anti-HER2 biparatopic ADCs as described herein, the method comprising incubating an anti-HER2 monospecific antibody or its antigen-binding fragment or an anti-HER2 biparatopic antibody of the present invention with a sample isolated from a patient, and detecting whether the antibody binds to the sample.

[0024] Other features and advantages of the present invention will become apparent from the detailed description of the following embodiments and drawings. However, the drawings and specific embodiments should not be construed as limiting the scope of the invention, and it should be understood that various changes and modifications within the spirit and scope of the invention, which will become apparent to those skilled in the art from this detailed description, are included within the scope of protection of the appended claims. The contents of all references cited herein, including publications, patents and published patent applications, are incorporated in their entirety by reference. [Brief explanation of the drawing]

[0025] [Figure 1] This study demonstrates the detection of the binding activity of anti-HER2 chimeric antibodies to human ErbB / HER family members EGFR, HER2, HER3, and HER4 by ELISA using control antibodies including trastuzumab, cetuximab, and patritumab.

[0026] [Figure 2] Using trastuzumab as a control antibody, we demonstrate the detection of the binding activity of an anti-HER2 chimeric antibody to the HER2-overexpressing cell line NCI-N87 by flow cytometry.

[0027] [Figure 3] Figure 3A shows the schematic structure of recombinant human-mouse chimeric proteins of the HER2 extracellular domain, and Figure 3B shows the results of an ELISA that detects the subdomains of the HER2 extracellular domain to which each anti-HER2 chimeric antibody binds.

[0028] [Figure 4] This shows the detection of binding competition between anti-HER2 chimeric antibodies or with the control antibody trastuzumab using competitive ELISA. Figure 4A shows the results for biotin-labeled mAb2164 competing with mAb2117, mAb2126, mAb2170, and trastuzumab for recombinant HER2 protein binding. Figure 4B shows the results for mAb2117 competing with biotin-labeled mAb2128 or biotin-labeled mAb2126 for recombinant HER2 protein binding, and for mAb2164 competing with biotin-labeled mAb2128 for recombinant HER2 protein binding.

[0029] [Figure 5] Using trastuzumab and pertuzumab as control antibodies, the effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on phosphorylation of the Y1248 site within the intracellular domain of HER2 in SKBR-3 cells are demonstrated.

[0030] [Figure 6] Using trastuzumab and pertuzumab as control antibodies, the effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on NRG-1-induced AKT phosphorylation in T47D cells are demonstrated.

[0031] [Figure 7] Using the parental scFv Hu2117HK as a control antibody, we demonstrate the detection of the optimized anti-HER2 scFv mutant protein's binding activity to BT474 cells (Figure 7A) and RT-112 cells (Figure 7B) by flow cytometry.

[0032] [Figure 8] Using the parental scFv Hu2117HK as a control antibody, we demonstrate the detection of the binding activity of optimized anti-HER2 scFv molecules containing single or multipoint mutations (including Hu2117-HK203, Hu2117-HK303, Hu2117-HK304, Hu2117-HK309, and Hu2117-HK310) to BT474 and RT-112 cells by flow cytometry.

[0033] [Figure 9] The schematic structure of a representative anti-HER2 biparatopic antibody, 04BS-109-WT, is shown. This biparatopic antibody is constructed with a DVD-IgG configuration. The Fv domain contains the variable region sequence Hu2117-HK304, and the IgG domain contains the full-length sequence Hu2126-H2K1-L71-H72b-Mu14. The heavy chain variable region of the Fv domain is linked to the heavy chain of the IgG domain via linker (G4S)1, and the light chain variable region of the Fv domain is linked to the light chain of the IgG domain via linker (G4S)3. The heavy chain Fc region of the antibody contains site mutations L234F / L235E / P331S (EU numbering).

[0034] [Figure 10] This shows the detection of simultaneous or competitive binding between the humanized anti-HER2 antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) to BT474 cells by flow cytometry.

[0035] [Figure 11]This paper demonstrates the detection of the internalization dynamics of the anti-HER2 biparatopic antibody 04BS-109-WT and its variants in cells expressing different levels of HER2 (e.g., BT474, JIMT-1, RT-112) by flow cytometry, using a control antibody containing trastuzumab, as well as Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) (both monospecific antibodies were used to construct the biparatopic antibody 04BS-1123-ST06).

[0036] [Figure 12] This image shows the detection of internalization and lysosomal transport induced by the anti-HER2 biparatopic antibody 04BS-1123-ST06 in SKBR-3 cells using confocal microscopy with control antibodies including trastuzumab and human IgG isotype controls. The arrows indicate co-localization of the fluorescence signals of the antibody and lysosomes, demonstrating that HER2 aggregation during crosslinking with the biparatopic antibody is internalized and transported to lysosomes. T indicates the antibody incubation time (h).

[0037] [Figure 13] This shows the detection of HER2 proteolysis in BT474 cells after incubation by Western blotting, using a control antibody containing trastuzumab, as well as Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) (both monospecific antibodies used to construct the biparatopic antibody).

[0038] [Figure 14]The effect of the anti-HER2 biparatopic antibody 04BS-1123-ST06 on BT474 cell proliferation in vitro is demonstrated using control antibodies including trastuzumab and pertuzumab, as well as Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) (both monospecific antibodies were used to construct the biparatopic antibody).

[0039] [Figure 15] Using control antibodies containing trastuzumab and pertuzumab, the effects of anti-HER2 monospecific antibodies (Figure 15A) and anti-HER2 biparatopic antibodies (Figure 15B) on NRG-1-induced HER2 / HER4 dimerization by reporter gene assays are demonstrated.

[0040] [Figure 16] The effect of the anti-HER2 biparatopic antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes is demonstrated using control antibodies including trastuzumab and pertuzumab.

[0041] [Figure 17] Using trastuzumab as a positive control antibody, this report demonstrates the detection of ADCC activity of the anti-HER2 biparatopic antibody 04BS-1123-ST06 by reporter gene assay.

[0042] [Figure 18] This shows the detection of the binding specificity of anti-HER2 biparatopic ADC (ST06-VCP-eribulin) to human ErbB / HER family members (including EGFR, HER2, HER3, and HER4) using ELISA.

[0043] [Figure 19]This report demonstrates the detection of in vitro cytotoxic activity of anti-HER2 biparatopic ADCs (including ST06-GGFG-eribulin and ST06-VCP-eribulin) against a panel of cancer cell lines expressing different levels of HER2, compared to the benchmark molecule DS-8201.

[0044] [Figure 20] The bystander effect of anti-HER2 biparatopic ADCs detected by in vitro cell toxicology assays is shown. In Figure 20A, BT474 cells were treated with ST06-GGFG-eribulin for 3 days, and cytotoxicity was confirmed by in vitro cell toxicology assays. Meanwhile, the BT474 condition medium was collected and used to incubate with MDA-MB-468 cells for 3 days. The viability of MDA-MB-468 cells was measured by in vitro cell toxicology assays to detect the bystander toxicity effect, with the control group being MDA-MB-468 cells incubated in parallel with freshly prepared ST06-GGFG-eribulin. In Figure 20B, HER2-overexpressing BT474 cells and HER2-null Jurkat cells were treated with ST06-GGFG-eribulin in either monoculture or coculture, and cell viability was detected by flow cytometry. The numbers shown in the upper left and lower right corners represent the number of viable BT474 and Jurkat cells, respectively.

[0045] [Figure 21] This figure shows the detection of in vivo antitumor activity of anti-HER2 biparatopic ADC (ST06-GGFG-eribulin) in mouse subcutaneous xenograft tumor models established using tumor cell lines NCI-N87 (Figure 21A), JIMT-1 (Figure 21B), and RT-112 (Figure 21C). Controls included a mixture of antibody and small molecule compound (ADMix), DS-8201, and vehicle groups. All tumor-bearing mice were administered via intravenous injection, and arrows indicate administration time.

[0046] [Figure 22]This study demonstrates the detection of in vivo antitumor activity of an anti-HER2 biparatopic ADC (ST06-GGFG-eribulin) in a mouse subcutaneous xenograft tumor model with acquired resistance to DS-8201, with the control group containing DS-8201 and the vehicle. [Modes for carrying out the invention]

[0047] Definition: Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. For the purposes of the present invention, the following terms are defined below.

[0048] As used herein, “HER2” and “HER2 receptor” are interchangeable, and this protein is also referred to as ErbB2, c-ERB2, c-ERB-2, NEU, HER-2 / neu, p185(erbB2), or CD340. Unless expressly specified that it is derived from a non-human species (e.g., “mouse HER2,” “monkey HER2,” etc.), “HER2” as used herein refers to any native form of human HER2 that may have the amino acid sequence shown in SEQ ID NO: 234 and / or the full-length HER2 amino acid sequence shown in NCBI accession number NP_004439.2, and may be expressed naturally by cells (including tumor cells) or by cells transfected with the HER2 gene or cDNA. This term includes naturally occurring alleles and splice variants, isoforms, homologs, and species homologs of HER2. HER2 may be isolated from the human body or produced by recombinant or synthetic methods.

[0049] The HER2 extracellular domain consists of four subdomains: 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) (the numbering of residues excludes the signal peptide). Of these, 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).

[0050] As used herein, “HER2-expressing cells” may be naturally occurring cells or cell lines (e.g., tumor cells) or may be recombinantly produced by introducing the nucleic acid encoding HER2 into a host cell.

[0051] As used herein, “bispecific antibody” is intended to include any antibody or antigen-binding fragment that can specifically bind to two different epitopes (or antigenic determinants) and comprises two independent antigen-binding domains, each having its own unique antigen-binding specificity. For example, “biparatopic antibody” as used herein specifically refers to a class of bispecific antibodies in which a first antigen-binding domain and a second antigen-binding domain each bind to different epitopes on the same antigen.

[0052] A monospecific antibody refers to an antibody or antigen-binding fragment that has only one binding specificity, meaning that the antigen-binding domain of the monospecific antibody binds to a single epitope on a single antigen. In some embodiments, examples of monospecific antibodies include the anti-HER2 monospecific antibody of the present invention.

[0053] As used herein, “antigen-binding domain,” “antigen-binding region,” “epitope-binding domain,” or “antigen-binding polypeptide” may be used interchangeably and refer to a specific region on an antibody or its antigen-binding fragment or derivative that is directly involved in the interaction with the target antigen through a mechanism that achieves a dynamic equilibrium, such as binding, steric hindrance, stabilization / destabilization, or spatial distribution.

[0054] In the present invention, the "antigen-binding domain" also refers to a specific region on an antibody or its antigen-binding fragment or derivative that interacts with a specific epitope on HER2, and the interaction between the two achieves dynamic equilibrium through mechanisms such as binding, steric hindrance, stabilization / destabilization, and spatial distribution.

[0055] As used herein, “antibody” generally refers to a polypeptide or protein, encoded by one or more immunoglobulin genes or fragments thereof, that can specifically recognize and bind to an antigen. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, each defining an immunoglobulin class or isotype as IgG, IgM, IgA, IgD, and IgE. Some of these classes can be further divided into subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The typical structural unit of an immunoglobulin (e.g., an antibody) is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair containing a “light” chain (approximately 25 kDa) and a “heavy” chain (approximately 50–70 kDa). The N-terminus of each chain defines a variable (V) region, primarily responsible for antigen recognition, containing approximately 100–110 or more amino acids. The heavy chain of an antibody consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), the heavy chain constant region typically containing three domains: CH1, CH2, and CH3. The light chain consists of a light chain variable region (VL) and a light chain constant domain (CL), the light chain constant domain usually containing one domain CL. The pairing of VH and VL forms a single antigen-binding site. The endogenous VL is encoded by the V (variability) and J (linking) gene segments, and the endogenous VH is encoded by the V, D (variability), and J gene segments. The VL or VH contains a hypervariable region (i.e., a complementarity-determining region (CDR)) and a framework region (FR). The terms “variable region” or “V region” can be used interchangeably and refer to either a heavy-chain or light-chain variable region arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus. The term “J region” refers to a subsequence that codes for the C-terminal portion of the variable region, including CDR3 and FR4. The V region or J region may be naturally occurring, recombinant, or synthetic.As used herein, the light chain variable region and / or heavy chain variable region of an antibody may be collectively referred to as the “antibody variable region,” and the light chain and / or heavy chain of an antibody may be collectively referred to as the “antibody chain.” In specific embodiments, the FR of an antibody or its antigen-binding fragment provided herein may be identical to a human germline sequence or may be naturally or artificially modified.

[0056] The positions of CDR and FR are defined by various methods well known in the art, e.g., Kabat, Chothia, IMGT, and Contact (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 1991, Fifth Edition, NIH Publication No. 91-3242; Johnson et al., Nucleic Acids Research 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 MP et al., Nucleic Acids Research) This can be determined using (1999,27:209-212; MacCallum RM et al., J Mol Biol 1996,262:732-745). The definition of antigen-binding sites can also be found in Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefranc MP, Nucleic Acids Res 2001, 29:207-209; Lefranc MP, The Immunologist 1999, 7:132-136; Lefranc MP 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 MJE (edition), Protein Structure Prediction: A Practical This is described in Approach, 1996, Oxford University Press, pp. 141-172.The present invention includes any of the defining methods for determining the CDR in the anti-HER2 biparatopic antibody or anti-HER2 antibody or its antigen-binding fragment. Table 1 shows the amino acid residues of antibody CDRs determined by different defining methods. The exact number of amino acid residues contained in a particular CDR varies depending on the CDR sequence. If the amino acid sequence of the variable region of the antibody is known, those skilled in the art can determine the antibody CDR by conventional defining methods, including (but not limited to) the defining methods herein.

[0057] [Table 1]

[0058] Furthermore, Kabat et al. have defined a variable region sequence numbering system that can be applied to any antibody. Those skilled in the art will be able to 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 specified, the numbering of specific amino acid residue positions in the variable region of the antigen-binding domain of the anti-HER2 antibody, anti-HER2 biparatopic antibody, or biparatopic ADC described in the present invention is determined according to the Kabat numbering system.

[0059] Antibodies exist as intact immunoglobulins or as many well-characterized fragments produced by protease digestion. While various antibody fragments are defined based on the digestion of intact antibodies, those skilled in the art should understand that such fragments can be produced by either chemical cleavage or recombinant DNA methods. As used herein, the term “antigen-binding fragment” (or “antibody portion” or “antibody fragment”) of an antibody refers to a portion of an antibody containing one or more CDRs, or any other antibody fragment that can bind to an antigen (such as HER2 or the extracellular domain of HER2) without using the entire antibody structure. Antigen-binding fragments can exhibit the same antigen-specific binding activity as intact antibodies. Preferred antigen-binding fragments maintain the ability to be internalized into cells expressing the target antigen. In some embodiments, an antigen-binding fragment may comprise one or more CDRs derived from a particular human antibody, grafted onto one or more framework regions derived from different human antibodies. Antigen-binding fragments include, but are not limited to, the following: (i) "Fab" fragments, which are monovalent antibody fragments consisting of VH, VL, CL, and CH1 domains; (ii) "F(ab')2" fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds within a hinge region; (iii) "Fv" fragments, which are the smallest antibody fragments consisting of VL and VH domains derived from a single antibody arm and containing a complete antigen-binding site; (iv) "Fd" fragments, which consist of VH and CH1 domains; and (v) "single-chain Fv antibodies (scFv)" or "single-chain antibodies," which are engineered antibodies consisting of a light chain variable region linked directly to the heavy chain variable region or by a peptide chain (Huston et al., Proc Natl Acad Sci USA 1988, 85:5879-5883; Bird et al., Science 1988, 242:423-426). These scFv antibodies can be internalized into cells after binding to antigens on the cell surface (He et al., J Nucl Med 2010, 51:427-432; Fitting et al., MAbs 2015, 7:390-402).Furthermore, single-chain antibodies include "linear antibodies" that contain a tandem Fv segment (VH-CH1-VH-CH1) that forms a pair of antigen-binding domains when paired with a complementary light chain polypeptide (Zapata et al., Protein Eng 1995, 8:1057-1062; US5641870). (vi) "dAb" fragments containing a single variable domain, such as a VH domain (Ward et al., Nature 1989, 341:544-546; WO90 / 05144A1). Single-domain antibodies (sdAb) are independent immunoglobulin domains. (vii) "Diabodies" are bivalent, bispecific antibodies. In this antibody, the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short for the two domains on the same chain to pair with each other. Therefore, they are paired with the complementary domain of the other 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; EP404097; WO93 / 11161).

[0060] As used herein, the terms “Fc region” or “Fc domain” refer to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of a constant region, such as the immunoglobulin heavy chain constant region excluding the first constant region (CH1). In the case of IgG, the Fc region may include the immunoglobulin domains CH2 and CH3, as well as the hinge region between CH1 and CH2. As used herein, the Fc region includes the natural Fc region and / or Fc region variants, and may be part of the anti-HER2 antibody, anti-HER2 biparatopic antibody or its ADC described in the present invention. While the boundaries of the Fc region can vary, it will be understood in the art that the human IgG heavy chain Fc region is generally defined as containing either the cysteine ​​residue at position 226 or the proline residue at position 230 at its amino terminus, according to the EU numbering system / scheme described in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5th edition, NIH Publication No. 91-3242.

[0061] The terms “anti-HER2 antibody” or “HER2-specific antibody” refer to any form of antibody or fragment that specifically binds to HER2, and include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as the fragment specifically binds to HER2.

[0062] In this specification, the terms “specifically binding,” “binding specificity,” “specific to,” or “binding” refer to a binding reaction that determines the presence of a target molecule (or protein or antigen) in a heterogeneous population of proteins and other biological materials (e.g., biological samples such as blood, serum, plasma, or tissue samples). That is, binding is selective to the target molecule and can be distinguished from their undesirable or nonspecific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) exhibits higher affinity, stronger binding activity, easier binding, and / or longer binding duration compared to binding to other non-target molecules. Therefore, if necessary, such selection can be achieved by excluding antibodies that cross-react with other members of the ErbB / HER family. Various immunoassays, such as ELISA, can be used to select antibodies that specifically react with a particular protein. Typically, under defined assay conditions, the specific or selective binding reaction of the antibody or binder to its antigen should produce a signal at least twice as loud as the background level, more commonly at least 10 to 100 times louder than the background, with substantially no significant binding to other antigens present in the sample. In some embodiments, the antibody has a dissociation equilibrium constant (K) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. D It binds specifically to the target antigen at a value of ).

[0063] In this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for any minor native variations that may be present. Monoclonal antibodies exhibit high binding specificity and affinity for specific epitopes. Monoclonal antibodies can be produced by the hybridoma method, first described by Kohler et al., Nature 1975, 256:495, or by the recombinant DNA method (see US4816567), and can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al., Nature 1991, 352:624-628; Marks et al., J Mol Biol 1991, 222:581-597.

[0064] In this specification, the term “chimeric antibody” refers to an antibody containing sequences derived from two different antibodies (e.g., US4,816,567), where the antibodies are typically derived from different species. For example, a chimeric antibody may contain human and rodent antibody fragments, usually a human constant region and a mouse variable region. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those skilled in the art (e.g., Morrison SL et al., Proc Natl Acad Sci USA 1984, 81:6851-6855; US5202238 and US5204244).

[0065] In this specification, the term “humanized antibody or its antigen-binding fragment” refers to an antibody or its antigen-binding fragment comprising a CDR derived from a non-human animal, a human-derived FR region, and a human-derived constant region. A humanized antibody may optionally further comprise at least a portion of the constant region of a human immunoglobulin. Because of their reduced immunogenicity, humanized antibodies or their antigen-binding fragments can be used as therapeutic agents for 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, the humanized antibody or its antigen-binding fragment consists of an entirely human sequence, except for the non-human CDR sequence. In some embodiments, the humanized antibody can be further modified, improved, and optimized by substituting residues in the FR of the human immunoglobulin with corresponding residues derived from a non-human species antibody to enhance their specificity, affinity, and / or activity. In some embodiments, the human-derived FR may comprise the same amino acid sequence as the human antibody from which it originates, or may comprise several amino acid mutations, e.g., 5, 4, 3, 2, or 1 or fewer amino acid modifications. In some embodiments, amino acid modifications may be present only in the heavy chain FR, only in the light chain FR, or in both chains.

[0066] In this specification, the term “corresponding human germline sequence” refers to an antibody variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with the reference human germline immunoglobulin variable region amino acid sequence compared to all other known human germline immunoglobulin variable region amino acid sequences. The corresponding human germline sequence may simply be a framework region, a complementarity-determining region, a framework region and a complementarity-determining region, a variable region, or any other combination including a variable region sequence or subsequence. Sequence identity can be determined by aligning the two sequences using the methods described herein, for example, BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the reference human germline immunoglobulin variable region amino acid sequence.

[0067] The antibody component of the anti-HER2 antibody, anti-HER2 biparatopic antibody, or ADC of the present invention can be selected from one or more of the following forms, as long as it is in a form that can specifically bind to HER2, and includes chimeric, non-human, humanized, or fully human forms.

[0068] As used herein, the term “epitope” refers to a protein determinant, which is a part of an antigen that can be specifically recognized and bound by an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally possess specific three-dimensional structural and specific charge properties. The portion of an antibody or its antigen-binding fragment that recognizes an epitope is called a paratope.

[0069] Using competitive binding and epitope binning, it is possible to determine whether different antibodies bind to the same or overlapping epitopes, such as by the method described in Harlow and Lane (eds.), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory for evaluation. In some embodiments, competitive binding ELISA is used to evaluate whether an antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment containing a CDR and / or variable region as defined in Tables 2 and 3) inhibits the binding activity of another antibody or antigen-binding fragment against a target antigen (e.g., HER2). If 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 listed values), it is determined that the antibodies bind competitively, i.e., to the same or overlapping epitopes. In some embodiments, competitive binding may result from steric hindrance caused by antibodies or antigen-binding fragments binding to covalent or similar epitopes (e.g., partially overlapping) or adjacent 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 covalent / similar epitopes. For example, antibodies or antigen-binding fragments exhibiting competitive binding may be "binned" into a group of antibodies or antigen-binding fragments that target overlapping or adjacent epitopes, while non-competitive antibodies or antigen-binding fragments may be classified into a separate group that targets non-overlapping or non-adjacent epitopes.

[0070] 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 specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., receptor and ligand, or antigen and antibody). The affinity of molecule X for its binding partner Y is usually expressed by the dissociation rate constant (k dis or k off ) and coupling rate constant (k a or k on The dissociation equilibrium constant (K) is the ratio of ) D ) can be expressed as. Affinity can be measured by conventional methods known in the art, including those used in the present invention.

[0071] As used herein, “internalization” or “endocytosis” refers to the process by which an antibody or its antigen-binding fragment, bispecific antibody, or ADC can enter the cell’s endosomes by internalization (i.e., endocytosis) across the cellular lipid bilayer membrane after binding to a target antigen on the cell surface, preferably the transport of the antibody or antigen-binding fragment, or bispecific antibody, into intracellular lysosomes. In some embodiments, the anti-HER2 biparatopic antibody or ADC described in the present invention can be internalized into the cell after binding to HER2 on the tumor cell membrane, thereby downregulating HER2 expression on the tumor cell surface.

[0072] As used herein, the terms “antibody variant” or “antibody variant” refer to an antibody polypeptide sequence having at least one amino acid mutation in the variable region of the original antibody. The variant may be substantially homologous or substantially identical to the unmodified antibody. In some embodiments, introducing amino acid mutations into one, two, three, four, five and / or six CDRs of the antibody component of the anti-HER2 antibody, anti-HER2 biparatopic antibody, or ADC of the present invention improves and optimizes the performance of the antibody or antigen-binding fragment, including, but is not limited to, an increased degree of humanization, enhanced binding affinity or binding activity to HER2, improved internalization efficiency, increased production yield, and / or improved stability (e.g., reduced or eliminated risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, the antibody component of the anti-HER2 antibody, anti-HER2 biparatopic antibody, or ADC of the present invention is improved and optimized in performance by including amino acid mutations in 1, 2, 3, and / or 4 FRs, for example, by increasing the degree of humanization of the antibody or antigen-binding fragment, increasing binding affinity or activity to HER2, increasing production yield, and / or improving stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, the degree of humanization of the antibody or antigen-binding fragment is increased by introducing one or more amino acid mutations in both the CDR and FR of the antibody component of the anti-HER2 antibody, anti-HER2 biparatopic antibody, or ADC of the present invention, thereby enhancing binding affinity or activity to HER2, improving internalization efficiency, increasing production yield, and / or improving stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, amino acid mutations include amino acid substitutions, deletions, insertions, or any combination thereof.

[0073] Of these, amino acid substitutions include conserved and non-conserved amino acid substitutions. Conserved substitutions relate to substituting one amino acid of the same class (e.g., similar biochemical properties or functions) with another, while non-conserved substitutions relate to substituting one amino acid of one class with another amino acid of a different class (different chemical properties or functions). A person skilled in the art can perform conserved or non-conserved amino acid substitutions based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, (i) nonpolar (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); and (iv) positively charged (acidic) amino acids include aspartic acid (Asp,D) and glutamic acid (Glu,E). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein, while non-conservative amino acid substitutions can lead to significant changes in the properties or function of a protein. While the sites or regions where amino acid mutations are introduced can be predetermined, the potential changes in protein properties or function caused by non-conservative substitutions are unpredictable.

[0074] The terms “identical,” “identity,” “identity percentage,” or “sequence identity percentage” as used herein may be used interchangeably for two or more polypeptide sequences and refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence after the amino acid sequences have been aligned and gaps introduced as necessary to maximize the number of identical amino acids. Conservative substitutions of such amino acid residues may or may not be considered identical residues. The sequence identity percentage of an amino acid sequence can be determined by aligning the sequences using tools disclosed in the art, such as BLASTp, ClustalW2 (see, e.g., Higgins et al., Methods in Enzymology 1996, 266:383-402; Larkin et al., Bioinformatics 2007, 23:2947-2948) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can appropriately adjust the parameters according to the alignment requirements by using the tool's default parameters or by selecting an algorithm suitable for array alignment.

[0075] In this specification, when referring to proteins, the term “isolated” means that the protein is substantially free of other cellular components bound in its native state and is preferably homogeneous; for example, an isolated protein may be taken from its native or natural environment. An isolated protein may be a lyophilized product or an aqueous solution. Generally, its purity and homogeneity can be determined by using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. As a main component in a formulation, the protein is essentially obtained by purification. The term “purified” means that the protein produces substantially one band on an electrophoretic gel. In particular, it means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. In some embodiments of the present invention, recombinant proteins expressed in host cells are considered isolated, but this applies to native or recombinant proteins that have been separated, fractionated, or partially or substantially purified by any technique well known to those skilled in the art. In some embodiments, “isolated antibody” refers to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to HER2 substantially does not contain other antibodies that specifically bind to antigens other than HER2). However, an isolated antibody that specifically binds to HER2 may cross-react with other antigens, such as HER2 proteins from other species, such as monkey HER2. Furthermore, an isolated antibody may not substantially contain other cellular material and / or chemicals. In some embodiments, a recombinant polynucleotide encoding the polypeptide or protein of the present invention (e.g., an anti-HER2 antibody, an anti-HER2 biparatopic antibody) contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells, or (partially or substantially) purified polynucleotides in solution.

[0076] The term “engineering” as used herein is interpreted to include any manipulation of polypeptides or protein backbones, or post-translational modifications of native or recombinant proteins or polypeptides. Engineering includes the introduction of mutations into amino acid sequences, modification of glycosylation patterns or amino acid side chains, and combinations thereof.

[0077] As used herein, the term “polypeptide” refers to polymers of amino acids and their equivalents, rather than products of a specific length; therefore, “peptides” and “proteins” are included within the definition of polypeptide. “Antibodies” as defined herein are also included within the definition of polypeptide.

[0078] Bivariable-domain immunoglobulin (DVD-Ig) is a tetravalent molecule containing two distinct antigen-binding domains, constructed by fusing the heavy chain variable region (VH) and light chain variable region (VL) domains of one antibody to the N-terminuses of the heavy and light chains of another IgG antibody, respectively. Thus, each half of DVD-Ig contains one heavy chain polypeptide and one light chain polypeptide, which, after dimerization, form two independent antigen-binding domains, one of which is the Fv fragment and the other the Fab fragment or IgG domain. DVD-Ig can be bispecific, meaning it can bind to two different epitopes on the same antigen, or to two different antigens.

[0079] In this specification, the terms “expression vector” or “vector” refer to a vehicle capable of operably inserting a protein-coding polynucleotide or nucleic acid and expressing a protein. A vector can be used to transform, transduce, or transfect a host cell, thereby enabling the expression of its genetic material components in the host cell.

[0080] In this specification, 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 “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not have the same nucleic acid content as the parent cells and may contain mutations. The present invention includes mutant offspring screened or selected from the initially transformed cells for the same function or biological activity.

[0081] The terms “subject,” “patient,” or “individual” as used herein are interchangeable and include, but are not limited to, mammals, e.g., humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammalian vertebrates, e.g., birds (e.g., chickens or ducks) or fish, as well as non-mammalian invertebrates. In some embodiments, the subjects and pharmaceutical compositions involved in the use or methods of the present invention are used to treat non-human animals (preventively and / or therapeutically).

[0082] As used herein, “to treat or cure” a disease or symptom means to alleviate the disease or symptom, to slow the rate at which the disease or symptom develops or manifests, to reduce the risk of developing the disease or symptom, or to delay the onset of symptoms associated with the disease or symptom, to reduce or terminate symptoms associated with the disease or symptom, to bring about a complete or partial recovery from the disease or symptom, to cure the disease or symptom, or any combination of the foregoing.

[0083] The term “therapeutic dose” or “effective dose” refers to a dose or concentration that is effective in achieving prevention or improvement of symptoms associated with a disease or disorder, and / or reduction of the severity of the disease or disorder, over a desired period of time. The therapeutic dose of the preparations, antibodies or antigen-binding fragments, ADCs, or compositions thereof of the present invention may vary depending on factors such as the disease state, the age, sex and weight of the individual, and the ability of the antibody portion of the antibody or ADC to produce the desired response in the individual. The therapeutic dose may also be considered such that the toxic or adverse effects of the preparation, antibody or antigen-binding fragment, ADC, or composition thereof are less than the therapeutic benefits. The term “effective dose” refers to a sufficient amount of active ingredient or drug to provide a clinical benefit, including, but not limited to, improvement, relief or reduction of symptoms of a disease, disorder or its associated symptoms, or delay or cessation of disease progression.

[0084] As used herein, “low-level HER2,” “low HER2 expression,” “low HER2 expression,” “HER2 low expression,” or “HER2 low-level expression” means that when paraffin-embedded tissue sections of tumor biopsy are measured for ErbB2 / HER2 protein staining intensity by an immunohistochemical (IHC) assay (e.g., HercepTest®), the IHC score is 1+ (faint / barely discernible membrane staining is detected on only a portion of the membrane in more than 10% of tumor cells), or the IHC score is 2. + If there is weak to moderate overall membrane staining observed in more than 10% of tumor cells, and the degree of HER2 gene amplification in tumor cells is further measured by fluorescence in situ hybridization (FISH) (e.g., Inform™ [Ventana] or PathVysion™ [Vysis]), a negative score, i.e., IHC2+ / FISH, is obtained. - This refers to cancer cells / tumors, subjects, or patients. Most HER2-low-expressing tumors have significantly higher HER2 expression levels than normal tissues. HER2 expression levels in normal tissues can be measured by any method available to those skilled in the art.

[0085] "HER2-negative," "HER2-expression-negative," or "expressing limited levels of HER2" refers to cancer cells / tumors, subjects, or patients that have low HER2 expression as defined above, or have an IHC score of 0 (no staining observed, or membrane staining present in less than 10% of tumor cells).

[0086] "HER2-positive," "HER2-expression-positive," or "HER2-overexpression" is indicated by an IHC score of 3. + This refers to cancer cells / tumors, subjects, or patients that exhibit strong intact membrane staining (observed in more than 10% of tumor cells). HER2 positivity is defined as an IHC score of 2. + This means having a positive score on a FISH assay (e.g., subtractive probe technology chromogenic in situ hybridization [SPoT-Light HER2 CISH] test, inform-dual ISH test), i.e., IHC2+ / FISH. + This includes cancer cells / tumors, subjects, or patients.

[0087] As used herein, the “bystander effect” or “bystander killing effect” of an ADC is mediated by a free membrane-permeable small molecule toxin (e.g., an anticancer drug) which is released by target molecule-positive (e.g., HER2-positive) cancer cells and passively diffuses into the tumor microenvironment, killing adjacent cells such as adjacent cancer cells that do not express the target molecule or express it at low levels (e.g., HER2-negative) and are therefore insensitive to the ADC.

[0088] As used herein, the terms “pharmaceutically acceptable” or “pharmaceutically acceptable” generally refer to the indicated carriers, vehicles, diluents, adjuvants and / or salts that are chemically and / or physically compatible with the other components in the formulation and physiologically compatible with the subject.

[0089] When used in combination with a number, the term "approximately" means to encompass a number within a range having a lower limit of 5% less than the specified number and an upper limit of 5% more than the specified number, or in one embodiment, a lower limit of 10% less than the specified number and an upper limit of 10% more than the specified number, or in another embodiment, a lower limit of 15% less than the specified number and an upper limit of 15% more than the specified number, or in another embodiment, a lower limit of 20% less than the specified number and an upper limit of 20% more than the specified number.

[0090] The term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0091] Where used herein, the terms “include,” “have,” “contain,” or “equip” can be used interchangeably to mean including elements, integers, or steps, but not excluding any other elements, integers, or steps. Where used herein, the terms “include,” “have,” “contain,” or “equip” also encompass situations consisting of the above elements, integers, or steps, unless otherwise indicated.

[0092] Where used herein, the term “optionally” indicates whether or not the subject it modifies exists; for example, “the kit optionally includes at least one additional anti-tumor agent” means that the kit may or may not include at least one additional anti-tumor agent.

[0093] Where used herein, “several embodiments,” “one embodiment,” “one specific embodiment,” or “specific embodiment,” or any combination thereof, indicates that certain features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned terms in various parts of this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0094] Unless the context explicitly indicates otherwise, singular terms encompass multiple referents, and vice versa.

[0095] All patents, patent applications, and other publications are expressly incorporated herein by reference for explanatory and disclosure purposes. These publications are provided solely because their disclosures date prior to the filing date of this application. All statements regarding the dates of these documents, or representations of the contents of these documents, are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates of these documents or the contents of these documents.

[0096] Various aspects of the present invention are described in more detail in the following sections.

[0097] 1. Anti-HER2 antibody or its antigen-binding fragment In one embodiment, the present invention provides an isolated anti-HER2 antibody or an antigen-binding fragment thereof that can specifically bind to the extracellular domain of HER2.

[0098] The anti-HER2 antibody or its antigen-binding fragment of the present invention can specifically bind to D1, D3 and / or D4 of the HER2 extracellular region, preferably D1 or D3. The anti-HER2 biparatopic antibody of the present invention can be constructed using the antigen-binding domains of two anti-HER2 monospecific antibodies, each capable of binding to a different epitope on the HER2 extracellular region, and the two monospecific antibodies do not exhibit competitive inhibition in antigen-competitive binding assays (i.e., the antigen-binding epitopes do not overlap).

[0099] In some embodiments, the anti-HER2 biparatopic antibody of the present invention can be constructed using the antigen-binding domains of two anti-HER2 monospecific antibodies, the two monospecific antibodies being able to simultaneously bind to different epitopes on the HER2 extracellular region. Furthermore, the two monospecific antibodies are able to simultaneously bind to any two epitopes on D1, D3, and D4 of the HER2 extracellular region. Preferably, they are able to simultaneously bind to D1 and D3 of the HER2 extracellular region. For example, the antigen-binding domain of the first anti-HER2 antibody binds to D3 of the HER2 extracellular region, and the antigen-binding domain of the second anti-HER2 antibody binds to D1 of the HER2 extracellular region.

[0100] Anti-HER2 antibodies or their antigen-binding fragments have strong binding activity to HER2-expressing tumor cells, including HER2-overexpressing tumor cells (e.g., breast cancer cell line SKBR-3, ductal carcinoma cell line BT474, gastric cancer cell line NCI-N87, and ovarian cancer cell line SKOV-3), tumor cells expressing intermediate levels of HER2 (e.g., breast cancer cell line JIMT-1), tumor cells expressing low levels of HER2 (e.g., human bladder cancer cell line RT-112, breast cancer cell lines ZR-75-1, and T47D), and / or tumor cells expressing limited levels of HER2 (e.g., breast cancer cell line MCF-7). The binding affinity (K) of the anti-HER2 antibody or its antigen-binding fragment to HER2 is measured. D The value is 5 × 10 -8 Less than M, preferably 1 × 10 -8Less than M, 5 x 10 -9 Less than M or 1 × 10 -9 Less than M, more preferably 1 × 10 -9 The M value is less than 1. The anti-HER2 antibody or its antigen-binding fragment does not cross-react with other members of the ErbB / HER family (including EGFR, HER3, and HER4).

[0101] The anti-HER2 antibody or its antigen-binding fragment does not interfere with the regulation of HER2 expressed on the cell surface and its downstream signaling 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 in the intracellular domain of HER2 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 ligand-dependent or ligand-independent HER2 dimerization (including HER2:HER4 dimerization and / or HER2:HER3 dimerization) and its downstream signaling pathways, and the ligand includes NRG-1 or heregulin.

[0102] The anti-HER2 antibody of the present invention may optionally contain F(ab')2, Fab, Fab', Fv, scFv, scFv-Fc, a single-domain antibody (sdAb), or an IgG type. The antibody of the present invention may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, and may be a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, or an antibody fragment, provided that the antibody specifically recognizes epitopes D1, D3 and / or D4 (preferably D1 and / or D3) in the extracellular region of HER2 and does not affect HER2 or its signaling pathway expressed on the surface of tumor cells. In some embodiments, the anti-HER2 antibody is selected from a mouse anti-human HER2 antibody and its humanized optimized antibody. In some embodiments, the anti-HER2 antibody contains scFv, scFv-Fc, a Fab fragment, and / or an IgG type. For example, an antibody that specifically binds to D3 of the HER2 extracellular domain may be in scFv or scFv-Fc format, and an antibody that specifically binds to D1 of the HER2 extracellular domain may be in Fab or IgG format. The Fab and scFv formats can be interconverted as needed, and the conversion methods are known in the art (see, for example, the method described in Zhou et al., Mol Cancer Ther 2012, 11:1167-1476).

[0103] In another embodiment, the present invention provides an anti-HER2 antibody or antigen-binding fragment thereof comprising the CDR and / or variable region of an antibody shown in Table 2, which recognizes D3 of the HER2 extracellular domain, does not induce, block or inhibit phosphorylation and / or dephosphorylation of tyrosine residues in the HER2 intracellular domain, and / or does not induce, block or inhibit NRG-1-dependent or NRG-1-independent HER2 dimerization and its downstream signaling pathways. The present invention also provides an anti-HER2 antibody or antigen-binding fragment thereof capable of recognizing D1 of the HER2 extracellular domain, which does not induce, block or inhibit phosphorylation and / or dephosphorylation of tyrosine residues in the HER2 intracellular domain, and / or does not induce, block or inhibit NRG-1-dependent or NRG-1-independent HER2 dimerization and its downstream signaling pathways. Preferably, the antibody comprises the CDR and / or variable region of the antibody or antigen-binding fragment thereof shown in Table 3. The present invention also comprises a CDR, variable region, or light and heavy chains of the anti-HER2 antibody or antigen-binding fragment that recognizes D4 of the HER2 extracellular domain.

[0104] The heavy chain variable region (CDR) and light chain variable region (CDR) of the anti-HER2 antibody of the present invention are defined by the Kabat numbering system. However, as is known in the art, CDR regions can 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. All CDR regions defined by other numbering systems / methods, as well as CDR regions defined by Kabat as used in the present invention, are within the scope of protection of the present invention.

[0105] [Table 2-1] [Table 2-2]

[0106] [Table 3-1] [Table 3-2]

[0107] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment comprises VH having the amino acid sequence shown in SEQ ID NO: 1 and / or VL having the amino acid sequence shown in SEQ ID NO: 2, and can specifically bind to D3 of the HER2 extracellular domain.

[0108] In some embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention comprises VH having the amino acid sequence shown in SEQ ID NO: 3, 7, or 9, and / or VL having the amino acid sequence shown in SEQ ID NO: 4, 8, or 10, and can specifically bind to D1 of the HER2 extracellular domain. In one embodiment, the anti-HER2 antibody or antigen-binding fragment of the present invention that specifically binds to D1 of the HER2 extracellular domain comprises VH having the amino acid sequence shown in SEQ ID NO: 3, and / or VL having the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the anti-HER2 antibody or antigen-binding fragment of the present invention that specifically binds to D1 of the HER2 extracellular domain comprises VH having the amino acid sequence shown in SEQ ID NO: 7, and / or VL having the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the anti-HER2 antibody or antigen-binding fragment of the present invention that specifically binds to D1 of the HER2 extracellular domain comprises VH having the amino acid sequence shown in SEQ ID NO: 9, and / or VL having the amino acid sequence shown in SEQ ID NO: 10.

[0109] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment comprises VH having the amino acid sequence shown in SEQ ID NO: 5 and / or VL having the amino acid sequence shown in SEQ ID NO: 6, and can specifically bind to D4 of the HER2 extracellular domain.

[0110] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D3 of the HER2 extracellular domain comprises one or more CDRs of VH having the amino acid sequence shown in SEQ ID NO: 1, or comprises the amino acid sequences shown in SEQ ID NOs: 11, 14, and 32 or their variants, the variants of which include humanized antibodies or any other variants described herein.

[0111] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D3 of the HER2 extracellular domain further comprises one or more CDRs of a VL having the amino acid sequence shown in SEQ ID NO: 2, or comprises the amino acid sequences shown in SEQ ID NOs: 38, 46, and 49 or their variants, the variants of which include humanized antibodies or any other variants described herein.

[0112] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment includes HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 32, as well as LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 46, and LCDR3 shown in SEQ ID NO: 49.

[0113] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D1 of the HER2 extracellular domain comprises one or more CDRs of VH having the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequences shown in SEQ ID NOs: 98, 100, and 110, or a variant thereof, and / or comprises one or more CDRs of VH having the amino acid sequence shown in SEQ ID NO: 7, or the amino acid sequences shown in SEQ ID NOs: 205, 206, and 207, or a variant thereof, and / or comprises one or more CDRs of VH having the amino acid sequence shown in SEQ ID NO: 9, or the amino acid sequences shown in SEQ ID NOs: 211, 212, and 213, or a variant thereof, wherein the variants include humanized antibodies or any other variants described herein.

[0114] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D1 of the HER2 extracellular domain further comprises one or more CDRs of a VL having the amino acid sequence shown in SEQ ID NO: 4, or the amino acid sequences shown in SEQ ID NOs: 114, 116, and 139, or a variant thereof, and / or comprises one or more CDRs of a VL having the amino acid sequence shown in SEQ ID NO: 8, or the amino acid sequences shown in SEQ ID NOs: 208, 209, and 210, or a variant thereof, and / or comprises one or more CDRs of a VL having the amino acid sequence shown in SEQ ID NO: 10, or the amino acid sequences shown in SEQ ID NOs: 214, 215, and 216, or a variant thereof, wherein the variants include the humanized antibodies described herein or any other variants.

[0115] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D1 of the HER2 extracellular domain includes HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 100, HCDR3 shown in SEQ ID NO: 110, and LCDR1 shown in SEQ ID NO: 114, LCDR2 shown in SEQ ID NO: 116, and LCDR3 shown in SEQ ID NO: 139; or includes HCDR1 shown in SEQ ID NO: 205, HCDR2 shown in SEQ ID NO: 206, HCDR3 shown in SEQ ID NO: 207, and LCDR1 shown in SEQ ID NO: 208, LCDR2 shown in SEQ ID NO: 209, and LCDR3 shown in SEQ ID NO: 210; or includes HCDR1 shown in SEQ ID NO: 211, HCDR2 shown in SEQ ID NO: 212, HCDR3 shown in SEQ ID NO: 213, and LCDR1 shown in SEQ ID NO: 214, LCDR2 shown in SEQ ID NO: 215, and LCDR3 shown in SEQ ID NO: 216.

[0116] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D4 of the HER2 extracellular domain comprises one or more CDRs of VH having the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequences shown in SEQ ID NOs: 199, 200, and 201, or a variant thereof, the variants of which include humanized antibodies or any other variants described herein.

[0117] In some embodiments, the anti-HER2 antibody of the present invention or its antigen-binding fragment that specifically binds to D4 of the HER2 extracellular domain comprises one or more CDRs of a VL having the amino acid sequence shown in SEQ ID NO: 6, or the amino acid sequences shown in SEQ ID NOs: 202, 203, and 204, or a variant thereof, the variants of which include humanized antibodies or any other variants described herein.

[0118] In some embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention that specifically binds to D4 of the HER2 extracellular domain includes HCDR1 shown in SEQ ID NO: 199, HCDR2 shown in SEQ ID NO: 200, HCDR3 shown in SEQ ID NO: 201, and LCDR1 shown in SEQ ID NO: 202, LCDR2 shown in SEQ ID NO: 203, and LCDR3 shown in SEQ ID NO: 204.

[0119] In another embodiment, the VH and / or VL of the anti-HER2 antibody or antigen-binding fragment of the present invention can be used as a starting material for manipulating to produce the antibody described herein that is more suitable for administration to humans. The antibody can be manipulated by mutating one or more amino acid residues in one or both variable regions (i.e., VH and / or VL), for example, by mutating one or more amino acid residues in one or more CDR regions and / or one or more framework regions.

[0120] In some embodiments, the variable region of the anti-HER2 antibody of the present invention is manipulated by CDR grafting. The antibody interacts with the target antigen primarily through amino acid residues of six CDRs in the heavy and light chains. Therefore, the amino acid sequences within the CDR region of individual antibodies are more diverse than sequences outside the CDR region, such as FRs. Because the amino acid sequence of the CDR region is responsible for most of the antibody-antigen interaction, recombinant antibodies can be expressed to mimic the properties of a particular native antibody by constructing an expression vector containing the CDR sequence from that particular native antibody grafted onto the FR sequence of another antibody with different properties (see, for example, 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, as well as US5225539, US5530101, US5585089, US5693762 and US6180370).

[0121] The anti-HER2 antibodies of the present invention may also include different framework region sequences. Such framework region sequences can be obtained from publicly available DNA databases or publicly available references relating to germline antibody gene sequences. For example, germline DNA sequences of human heavy chain variable region genes and human light chain variable region genes can be found in the "V-Base" human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 1991, 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 which are explicitly incorporated herein by reference. As another example, germline DNA sequences of human heavy chain variable region genes 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 numbers: 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 numbers: IGKV1-39 or IGKV1-39*01.

[0122] The amino acid sequence of an antibody can be aligned based on the translated protein sequence using one of the sequence similarity search methods known to those skilled in the art, such as Gapped BLAST (Altschul et al., Nucleic Acids Res 1997, 25:3389-3402).

[0123] A preferred framework sequence used in the anti-HER2 antibody of the present invention is an acceptor framework region that is structurally similar (or highly homologous) to the framework sequence of the mouse parental antibody described in the present invention. In some embodiments, the CDR1, CDR2, and CDR3 region sequences of either VH or VL can be grafted onto an acceptor framework region, the acceptor framework region having identical or highest homologous sequences to the immunoglobulin gene in its corresponding germline. In specific embodiments, the present invention selects the CDR region of VH shown in SEQ ID NO: 1 or 3 and VL shown in SEQ ID NO: 2 or 4, and grafts it onto a human-derived IgG FR region to produce a humanized antibody, wherein the humanized antibody not only maintains antigen-binding activity similar to that of a parent antibody containing VH shown in SEQ ID NO: 1 and VL shown in SEQ ID NO: 2, or VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4, but also does not interfere with the HER2 downstream signaling pathway, for example, not affecting phosphorylation of tyrosine residues in the intracellular domain of HER2 (e.g., Y1248 phosphorylation), and / or ligand (e.g., NRG-1 or heregulin)-induced HER2 dimerization and the downstream signaling pathways mediated thereby.

[0124] Furthermore, the VH and VL sequences (or CDR sequences, or full-length heavy and 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 and light chain sequences) of the anti-HER2 antibody of the present invention. Preferably, when the VH and VL chains (or CDRs, or full-length heavy and light chain sequences within these chains) are mixed and matched, the VH sequence from a particular VH / VL pair is replaced with a structurally similar VH sequence. Similarly, preferably, the VL sequence from a particular VH / VL pair is replaced with a structurally similar VL sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy / light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the full-length light chain sequence from a particular full-length heavy / light chain pair should be replaced with a structurally similar full-length light chain sequence.

[0125] Accordingly, in one embodiment, the antibody of the present invention or its antigen-binding fragment comprises (a) a heavy chain variable region comprising an amino acid sequence listed in Table 2 or Table 3, and (b) a light chain variable region comprising an amino acid sequence listed in Table 2 or Table 3, or a VL derived from another anti-HER2 antibody that specifically binds to domain D3 or D1 of the HER2 extracellular domain.

[0126] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises (a) a heavy chain variable region containing an amino acid sequence listed in Table 2 or Table 3, or the VH of another anti-HER2 antibody that specifically binds to D3 or D1 of the HER2 extracellular domain, and (b) a light chain variable region containing an amino acid sequence listed in Table 2 or Table 3.

[0127] In specific embodiments, the humanized antibody or antigen-binding fragment of the present invention contains a VH 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 to the sequence shown in SEQ ID NO: 52. The antibody or antigen-binding fragment specifically binds to D3 of the HER2 extracellular domain.

[0128] In specific embodiments, the humanized antibody or antigen-binding fragment of the present invention includes a VL 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 to the sequence shown in SEQ ID NO: 81. The antibody or antigen-binding fragment specifically binds to D3 of the HER2 extracellular domain.

[0129] In one embodiment, the humanized antibody of the present invention comprises VH 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 to the sequence shown in SEQ ID NO: 52, and VL 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 to the sequence shown in SEQ ID NO: 81. The antibody or its antigen-binding fragment specifically binds to D3 of the HER2 extracellular domain.

[0130] In specific embodiments, the humanized antibody or antigen-binding fragment of the present invention contains a VH 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 to the sequence shown in SEQ ID NO: 143 or 170. The antibody or antigen-binding fragment specifically binds to D1 of the HER2 extracellular domain.

[0131] In specific embodiments, the humanized antibody or antigen-binding fragment of the present invention includes a VL 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 to the sequence shown in SEQ ID NO: 171. The antibody or antigen-binding fragment specifically binds to D1 of the HER2 extracellular domain.

[0132] In one embodiment, the humanized antibody of the present invention comprises VH 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 to the sequence shown in SEQ ID NO: 143 or 170, and VL 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 to the sequence shown in SEQ ID NO: 171. The antibody or its antigen-binding fragment specifically binds to D1 of the HER2 extracellular domain.

[0133] In some embodiments, the CDR sequence can be grafted onto a framework region containing one or more mutations compared to the germline sequence. For example, introducing amino acid mutations within the framework region can maintain or enhance the antigen-binding ability of the antibody (see, for example, U.S. Patent Nos. 5,530101, 5,585089, 5,693762, and 6,180370). In some embodiments, grafting the CDR of the parental antibody of the present invention onto an acceptor framework region containing 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 its antigen-binding affinity.

[0134] In some specific embodiments, the FR region of the anti-HER2 humanized antibody of the present invention, which specifically binds to D3 of the HER2 extracellular domain, contains one or more amino acid mutations to improve the antigen-binding affinity of the humanized antibody. For example, one or more amino acid mutations can be introduced into the FR region of VH as shown in SEQ ID NO: 52, and further amino acid mutations such as a mutation in the serine residue at position 30 of HFR1 (corresponding to position 30 of VH as shown in SEQ ID NO: 52) can be introduced into HFR1, and / or one or more amino acid mutations can be introduced into the FR region of VL as shown in SEQ ID NO: 81, and further, one or more amino acid mutations can be introduced into LFR1, LFR2 and / or LFR3. Specifically, one or more amino acid mutations can be introduced into 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 VL as shown in SEQ ID NO: 81), and the threonine residue at position 29 of LFR3 (corresponding to position 85 of VL as shown in SEQ ID NO: 81).

[0135] In a specific embodiment, the anti-HER2 humanized antibody of the present invention, which specifically binds to D3 of the HER2 extracellular domain, contains the amino acid mutation S30N in the HFR region for VH shown in SEQ ID NO: 52, and / or contains one or more amino acid mutations from D2I, M4L, A43S, and T85V in the LFR region for VL shown in SEQ ID NO: 81, thereby significantly improving the antigen-binding affinity of the antibody.

[0136] In some specific embodiments, the FR region of the anti-HER2 humanized antibody of the present invention, which specifically binds to D3 in the extracellular domain of HER2, includes one or more amino acid mutations to improve the degree of humanization of the antibody of the present invention. For example, one or more amino acid mutations can be introduced into the FR region of VH as shown in SEQ ID NO: 52, and further, amino acid mutations such as an amino acid mutation at the tyrosine residue at position 29 of HFR1 and / or the alanine residue at position 14 of HFR2 (corresponding to position 49 in the amino acid sequence shown in SEQ ID NO: 52) can be introduced into HFR1 and / or HFR2, and / or one or more amino acid mutations can be introduced into the FR region of VL as shown in SEQ ID NO: 81, and further, amino acid mutations such as an amino acid mutation at the phenylalanine residue at position 31 of LFR3 (corresponding to position 87 in VL as shown in SEQ ID NO: 81) can be introduced into LFR3. Specifically, anti-HER2 humanized antibodies that specifically bind to D3 of the HER2 extracellular domain include the following amino acid mutations, namely Y29F and / or A49S in the HFR region for VH as shown in SEQ ID NO: 52, and / or the amino acid mutation F87Y in the LFR region for VL as shown in SEQ ID NO: 81, thereby increasing the degree of humanization of the antibody.

[0137] In specific embodiments, the FR region of the anti-HER2 humanized antibody of the present invention, which specifically binds to D1 of the HER2 extracellular domain, contains one or more amino acid mutations. For example, the degree of humanization of the anti-HER2 antibody of the present invention can be improved by introducing one or more amino acid mutations into the FR region of VH shown in SEQ ID NO: 143, or by introducing one or more amino acid mutations into HFR1 and / or HFR3. Preferably, one or more amino acid mutations are introduced at the serine residue at position 9 of HFR1, the valine residue at position 18, and / or at the valine residue at position 3 of HFR3 (corresponding to position 68 of VH in SEQ ID NO: 143), the phenylalanine residue at position 4 (corresponding to position 69 of VH in SEQ ID NO: 143), the leucine residue at position 6 (corresponding to position 71 of VH in SEQ ID NO: 143), the valine residue at position 10 (corresponding to position 75 of VH in SEQ ID NO: 143), the isoleucine residue at position 17 (corresponding to position 82 of VH in SEQ ID NO: 143), and the phenylalanine residue at position 29 (corresponding to position 91 of VH in SEQ ID NO: 143). To improve the degree of humanization of the anti-HER2 antibody of the present invention, one or more amino acid mutations can be introduced into the FR region of VL as shown in SEQ ID NO: 171. For example, an amino acid mutation can be introduced at the asparagine residue at position 15 of LFR2 (corresponding to position 49 of VL in SEQ ID NO: 171).

[0138] In specific embodiments, the anti-HER2 humanized antibody of the present invention that specifically binds to D1 of the HER2 extracellular domain contains one or more of the following amino acid mutations in the HFR region for VH shown 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 that specifically binds to D1 of the HER2 extracellular domain contains one or more of the following amino acid mutations in the HFR region for VH shown in SEQ ID NO: 143: V68T, F69I, L71R, V75K, and F91Y, which can significantly improve the degree of humanization of the antibody.

[0139] In some embodiments, the present invention introduces amino acid mutations into the VH and / or VL CDR regions of a humanized antibody to improve one or more properties of the antibody, such as improved degree of humanization, improved antigen-binding affinity, increased production yield, and improved stability (e.g., reduced risk of aspartate isomerization and / or asparagine deamidation). Site-directed mutagenesis or PCR mutagenesis can be performed, and the effect of the mutation on the functional properties of the antibody can be evaluated using in vitro or in vivo assays known in the art. The mutation may be an amino acid substitution, addition, or deletion, preferably an amino acid substitution. Specifically, one, two, three, four, five, six, seven, eight, nine, or up to ten amino acid residues in the heavy chain CDR region or light chain CDR region are mutated.

[0140] In some specific embodiments, the anti-HER2 humanized antibody of the present invention that specifically binds to D3 of the HER2 extracellular domain contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations in the heavy chain CDR region or the light chain CDR region.

[0141] In a specific embodiment, one or more amino acid mutations can be introduced into the CDR region of VH as shown in Sequence ID No. 52, and further, one, two, three, four, or five amino acid mutations can be introduced into HCDR1, HCDR2, and / or HCDR3. For example, one or more amino acid mutations can be introduced into the serine residue at position 1 of HCDR1 (corresponding to position 31 of VH in SEQ ID NO: 52), the glycine residue at position 5 of HCDR2 (corresponding to position 53 of VH in SEQ ID NO: 52), the serine residue at position 7 of HCDR2 (corresponding to position 55 of VH in SEQ ID NO: 52), the threonine residue at position 9 of HCDR2 (corresponding to position 57 of VH in SEQ ID NO: 52), the proline residue at position 12 of HCDR2 (corresponding to position 60 of VH in SEQ ID NO: 52), the aspartic acid residue at position 13 of HCDR2 (corresponding to position 61 of VH in SEQ ID NO: 52), the serine residue at position 14 of HCDR2 (corresponding to position 62 of VH in SEQ ID NO: 52), and / or the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH in SEQ ID NO: 52).

[0142] In one embodiment, one or more amino acid mutations are introduced into HCDR1, HCDR2, and / or HCDR3 of VH as shown in SEQ ID NO: 52. For example, as shown in SEQ ID NO: 52, amino acid mutations are introduced into the serine residue at position 7 of HCDR2 of VH (corresponding to position 55 of VH as shown in SEQ ID NO: 52), the threonine residue at position 9 (corresponding to position 57 of VH as shown in SEQ ID NO: 52), the proline residue at position 12 (corresponding to position 60 of VH as shown in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 of VH as shown in SEQ ID NO: 52), the serine residue at position 14 (corresponding to position 62 of VH as shown in SEQ ID NO: 52), and / or the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH as shown in SEQ ID NO: 52).

[0143] In specific embodiments, one or more amino acid mutations can be introduced into the CDR region of the VL shown in SEQ ID NO: 81, and further, one, two, or three amino acid mutations can be introduced into LCDR1, LCDR2, and / or LCDR3. For example, one or more amino acid mutations can be introduced into the valine residue at position 7 (corresponding to position 27c of the VL shown in SEQ ID NO: 81), the histidine residue at position 8 (corresponding to position 27d of the VL shown in SEQ ID NO: 81), the glycine residue at position 11 of LCDR1 (corresponding to position 29 of the VL shown in SEQ ID NO: 85), the phenylalanine residue at position 6 of LCDR2 (corresponding to position 55 of the VL shown in SEQ ID NO: 81), and / or the serine residue at position 1 of LCDR3 (corresponding to position 89 of the VL shown in SEQ ID NO: 81), or the tyrosine residue at position 8 (corresponding to position 96 of the VL shown in SEQ ID NO: 81).

[0144] In one embodiment, the present invention introduces multiple amino acid mutations into the VH and VL CDR regions of the above-mentioned anti-HER2 humanized antibody that specifically binds to D3 of the HER2 extracellular domain, thereby increasing the degree of humanization of the antibody, increasing production yield, and / or improving stability (e.g., reducing the risk of aspartate isomerization). Specifically, one or more amino acid mutations are introduced into the serine residue at position 7 of HCDR2 in VH shown in SEQ ID NO: 52 (corresponding to position 55 in VH shown in SEQ ID NO: 52), the threonine residue at position 9 (corresponding to position 57 in VH shown in SEQ ID NO: 52), the proline residue at position 12 (corresponding to position 60 in VH shown in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 in VH shown in SEQ ID NO: 52), the serine residue at position 14 (corresponding to position 62 in VH shown in SEQ ID NO: 52), the alanine residue at position 3 of HCDR3 (corresponding to position 97 in VH shown in SEQ ID NO: 52), and the glycine residue at position 11 of LCDR1 in VL shown in SEQ ID NO: 81 (corresponding to position 29 in VL shown in SEQ ID NO: 81). Preferably, the amino acid mutation is selectively introduced into one or two of the following amino acid residues: the proline residue at position 12 of HCDR2 in VH shown in SEQ ID NO: 52 (corresponding to position 60 in VH shown in SEQ ID NO: 52), the aspartic acid residue at position 13 (corresponding to position 61 in VH shown in SEQ ID NO: 52), and the serine residue at position 14 (corresponding to position 62 in VH shown in SEQ ID NO: 52), as well as the alanine residue at position 3 of HCDR3 (corresponding to position 97 in VH shown in SEQ ID NO: 52), and the glycine residue at position 11 of LCDR1 in VL shown in SEQ ID NO: 81 (corresponding to position 29 in VL shown in SEQ ID NO: 81).

[0145] In some specific embodiments, the heavy chain CDR region or light chain CDR region of the anti-HER2 humanized antibody of the present invention, which specifically binds to D1 of the HER2 extracellular domain, contains 1, 2, 3, 4, or 5 or fewer amino acid mutations.

[0146] In a specific embodiment, one or more amino acid mutations can be introduced into the CDR region of VH as shown in Sequence ID No. 143, and further, one, two, three, four, or five amino acid mutations can be introduced into HCDR1, HCDR2, and / or HCDR3. For example, one or more mutations can be introduced into the serine residue at position 3 of HCDR1 (corresponding to position 33 of VH in SEQ ID NO: 143), the glutamic acid residue at position 5 of HCDR2 (corresponding to position 53 of VH in SEQ ID NO: 143), the glutamic acid residue at position 8 (corresponding to position 56 of VH in SEQ ID NO: 143), the aspartic acid residue at position 14 (corresponding to position 62 of VH in SEQ ID NO: 143), the phenylalanine residue at position 15 (corresponding to position 63 of VH in SEQ ID NO: 143), and / or the arginine residue at position 3 of HCDR3 (corresponding to position 97 of VH in SEQ ID NO: 143), the tyrosine residue at position 4 (corresponding to position 98 of VH in SEQ ID NO: 143), and the aspartic acid residue at position 5 (corresponding to position 99 of VH in SEQ ID NO: 143).

[0147] In a specific embodiment, one or more amino acid mutations are introduced into the CDR region of the VL shown in SEQ ID NO: 171. For example, mutations can be introduced into one or more of the following positions: the lysine residue at position 1 of LCDR1 (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 / or the histidine residue at position 3 of LCDR3 (corresponding to position 91 of the VL shown in SEQ ID NO: 171).

[0148] In one embodiment, the present invention introduces multiple amino acid mutations into the CDR regions of the VH and VL of the above-mentioned anti-HER2 humanized antibody that specifically binds to D1 of the HER2 extracellular domain, thereby improving the degree of humanization of the antibody, increasing antigen-binding affinity, increasing production yield, and / or improving stability (e.g., reducing the risk of potential asparagine deamidation). Specifically, mutations are introduced at the positions of the glutamic acid residue at position 5 of HCDR2 in VH shown in SEQ ID NO: 143 (corresponding to position 53 in VH shown in SEQ ID NO: 143), the lysine residue at position 1 of LCDR1 in VL shown in SEQ ID NO: 171 (corresponding to position 24 in VL shown in SEQ ID NO: 171), the serine residue at position 1 of LCDR2 (corresponding to position 50 in VL shown in SEQ ID NO: 171), the tyrosine residue at position 4 (corresponding to position 53 in VL shown in SEQ ID NO: 171), the tyrosine residue at position 6 (corresponding to position 55 in VL shown in SEQ ID NO: 171), and the histidine residue at position 3 of LCDR3 (corresponding to position 91 in VL shown in SEQ ID NO: 171).

[0149] In some embodiments, multiple amino acid mutations can be introduced into the CDR and framework regions of the variable domain 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, improving stability, or increasing production yield). In a specific embodiment, multiple amino acid mutations are introduced into one or two CDR regions and one HFR region of the VH shown in SEQ ID NO: 143, and / or three CDR regions of the VL shown in SEQ ID NO: 171, of the humanized antibody that specifically binds to D1 of the HER2 extracellular domain. Preferably, multiple amino acid mutations are present at the glutamic acid residue at position 5 of HCDR2 in VH shown in SEQ ID NO: 143 (corresponding to position 53 in VH shown in SEQ ID NO: 143), the valine residue at position 3 of HFR3 (corresponding to position 68 in VH shown in SEQ ID NO: 143), the phenylalanine residue at position 4 (corresponding to position 69 in VH shown in SEQ ID NO: 143), the leucine residue at position 6 (corresponding to position 71 in VH shown in SEQ ID NO: 143), the valine residue at position 10 (corresponding to position 75 in VH shown in SEQ ID NO: 143), and the phenylalanine residue at position 29 (sequence number The mutations are introduced at position 91 in VH shown in SEQ ID NO. 143, and / or at the lysine residue at position 1 of LCDR1 shown in SEQ ID NO. 171 (corresponding to position 24 in VL shown in SEQ ID NO. 171), the serine residue at position 1 of LCDR2 (corresponding to position 50 in VL shown in SEQ ID NO. 171), the tyrosine residue at position 4 (corresponding to position 53 in VL shown in SEQ ID NO. 171), the tyrosine residue at position 6 (corresponding to position 55 in VL shown in SEQ ID NO. 171), and the histidine residue at position 3 of LCDR3 (corresponding to position 91 in VL shown in SEQ ID NO. 171). These mutations not only further enhance the degree of humanization of the humanized antibody of the present invention, but also increase the binding affinity to HER2 (approximately 8 × 10⁻¹⁰). -9 ~9×10 -10 M, preferably 5 × 10 -9 ~9×10 -10 M's K D It significantly improves the value, reduces the risk of potential asparagine deamidation, and increases production yield.

[0150] 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 HER2 extracellular domain and comprises: (1) HCDR1 having the amino acid sequence X1YGMS (wherein X1=S, N, or D, i.e., the sequence shown in SEQ ID NO: 217, preferably X1=S); (2) SISGX2GX3YX4KYX5X6X7VKG (wherein X2=G or S, X3=S or N, X4=T) (3) HCDR2 having an amino acid sequence of 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 Sequence ID No. 218, preferably X2=G, X3=S, X4=T, X5=P or V, X6=D, P or E, X7=S or K), (4) HCDR3 having an amino acid sequence of DYX8GFFDV (wherein X8=A, I, N, R, S or V, i.e., the sequence shown in Sequence ID No. 219, preferably X8=V), (5) RSSQSLX9X 10 SNX 11 NTYLH(In the formula, X9 = V or L, X 10 =H or S, X 11 =G, A, I, S, R, or T, i.e., the sequence shown in sequence number 220, preferably X9=V or L, X 10 =H, X 11 LCDR1, (5)KVSNRX, which have the amino acid sequence =R) 12 S (where X 12 =F, D, or P, i.e., the sequence shown in Sequence ID No. 221, preferably X 12 LCDR2 having amino acid sequence =F), (6) amino acid sequence X 13 QSTHVPX 14 T(where, X 13 =S or Q, X 14 =Y or W, i.e., the sequence shown in sequence number 222, preferably X 13 =S, Christmas 14LCDR3 having (=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 above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The antibody or antigen-binding fragment exhibits high humanization, high production yield, and minimal aspartate isomerization risk.

[0151] In some specific embodiments, the antibody or antigen-binding fragments described above that specifically bind to D3 of the HER2 extracellular domain include: HCDR1 shown in SEQ ID NOs: 11, 12, or 13; HCDR2 shown in SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31; HCDR3 shown in SEQ ID NOs: 32, 33, 34, 35, 36, or 37; LCDR1 shown in SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, or 45; LCDR2 shown in SEQ ID NOs: 46, 47, or 48; and LCDR3 shown in SEQ ID NOs: 49, 50, or 51.

[0152] In specific embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention comprises 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 to the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and specifically binds to D3 of the HER2 extracellular domain: (1) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, 15, 16, 17, 18, 19 or 20, HCDR3 shown in SEQ ID NO: 32, and LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 46, LCDR3 shown in SEQ ID NO: 49, or (2) HCDR1 shown in sequence number 12 or 13, HCDR2 shown in sequence number 14, HCDR3 shown in sequence number 32, LCDR1 shown in sequence number 38, LCDR2 shown in sequence number 46, LCDR3 shown in sequence number 49, or (3) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 32, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 47 or 48, LCDR3 shown in SEQ ID NO: 49, or (4) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 32, LCDR1 shown in SEQ ID NOs: 39, 40, 41, 42, 43, 44 or 45, LCDR2 shown in SEQ ID NO: 46, LCDR3 shown in SEQ ID NO: 49, or (5) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 32, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 46, LCDR3 shown in SEQ ID NO: 50 or 51, or (6) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 33, 34, 35, 36 or 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 46, LCDR3 shown in SEQ ID NO: 49, or (7) HCDR1 shown in sequence number 11, HCDR2 shown in sequence numbers 14, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, HCDR3 shown in sequence number 37, LCDR1 shown in sequence number 42, LCDR2 shown in sequence number 46, LCDR3 shown in sequence number 49, or (8) HCDR1 shown in sequence number 11, HCDR2 shown in sequence number 21, HCDR3 shown in sequence number 32, LCDR1 shown in sequence number 42, LCDR2 shown in sequence number 46, and LCDR3 shown in sequence number 49.

[0153] In specific embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention specifically binds to D3 of the HER2 extracellular domain and contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. Preferably, the antibody or antigen-binding fragment contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. More preferably, the antibody or antigen-binding fragment contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0154] In other embodiments, the present invention provides an isolated anti-HER2 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to D1 of the HER2 extracellular domain and comprises: (1) DYX 15 MH (wherein, X 15 =S or A, i.e., the sequence shown in sequence number 223, preferably X 15 HCDR1 and (2)WINTX have the amino acid sequence =S). 16 TGX 17 PTYADX 18 X 19 KG (in the formula, 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 sequence shown in Sequence ID No. 224, preferably X 16 =G, X 17 =E, X 18 =D, X 19 HCDR2 and (3)VGX have the amino acid sequence =F). 20 X 21 X 22 YAMDY(in the formula, X 20 =R or Y, X 21 =Y or G, X 22 =D or S, i.e., the sequence shown in sequence number 225, preferably X 20 =R, X 21 =Y, X 22 HCDR3 having the amino acid sequence =D), (4)X 23 ASQDVYTAVA(where X 23 =K or R, i.e., the sequence shown in sequence number 226, preferably X 23 LCDR1, (5)X has the amino acid sequence =R) 24 ASX 25 RX 26 T(where, 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 Sequence ID No. 227, preferably X 24 =S, A, L or Y, X25 =S, X 26 LCDR2 having the amino acid sequence of =P), (6) the amino acid sequence QQX 27 YSTPPT (where X 27 =H, S, A or Y, that is, the sequence shown in SEQ ID NO: 228, preferably X 27 =Y) having LCDR3, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively. The antibody or antigen-binding fragment has a 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 also shows higher humanization and improved stability.

[0155] In a specific embodiment, the above antibody or antigen-binding fragment that specifically binds to D1 of the HER2 extracellular domain includes: HCDR1 shown in SEQ ID NO: 98 or 99, HCDR2 shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108 or 109, HCDR3 shown in SEQ ID NO: 110, 111, 112 or 113, LCDR1 shown in SEQ ID NO: 114 or 115, LCDR2 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 SEQ ID NO: 139, 140, 141 or 142.

[0156] In specific embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention comprises 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 to the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and specifically binds to D1 of the HER2 extracellular domain: (1) HCDR1 shown in sequence number 98, HCDR2 shown in sequence number 100, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 114, LCDR2 shown in sequence numbers 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128, LCDR3 shown in sequence number 139, or (2) HCDR1 shown in sequence number 98, HCDR2 shown in sequence number 100, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 114, LCDR2 shown in sequence number 116, LCDR3 shown in sequence number 140, 141 or 142, or (3) HCDR1 shown in sequence number 98, HCDR2 shown in sequence numbers 101, 102, 103, 104, 105, 106, 107, 108 or 109, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 114, LCDR2 shown in sequence number 116, LCDR3 shown in sequence number 139, or (4) HCDR1 shown in sequence number 98, HCDR2 shown in sequence number 100, HCDR3 shown in sequence number 111, 112 or 113, LCDR1 shown in sequence number 114, LCDR2 shown in sequence number 116, LCDR3 shown in sequence number 139, or (5) HCDR1 shown in sequence number 98, HCDR2 shown in sequence number 100, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 115, LCDR2 shown in sequence number 116, LCDR3 shown in sequence number 139, or (6) HCDR1 shown in sequence number 99, HCDR2 shown in sequence number 100, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 114, LCDR2 shown in sequence number 116, LCDR3 shown in sequence number 139, or (7) HCDR1 shown in sequence number 98, HCDR2 shown in sequence number 102, HCDR3 shown in sequence number 110, LCDR1 shown in sequence number 115, LCDR2 shown in sequence numbers 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138, and LCDR3 shown in sequence number 142.

[0157] In specific embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. Contains a mino acid sequence and can specifically bind to D1 of the HER2 extracellular domain: HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 100 or 102, HCDR3 shown in SEQ ID NO: 110, LCDR1 shown in SEQ ID NO: 114 or 115, LCDR2 shown in SEQ ID NOs: 116, 123, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138, and LCDR3 shown in SEQ ID NO: 139 or 142. Preferably, the antibody or antigen-binding fragment contains 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 with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, respectively.D ) is <5×10 -9 M, or <1 × 10 -9 M, preferably <1 × 10 -9 M is more preferably the antibody or its antigen-binding fragment contains 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 with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0158] In some embodiments, the anti-HER2 antibody or antigen-binding fragment of the present invention further comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3.

[0159] In specific embodiments, the antibody or antigen-binding fragment of the present invention can specifically bind to D3 of the HER2 extracellular domain, and it can bind to at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%) of the amino acid sequence shown in SEQ ID NOs. 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. It comprises a heavy chain variable region (VH) having at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs. 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97 (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%), and a light chain variable region (VL) having at least 80% identity (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%).

[0160] In specific embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, and is capable of specifically binding to D3 of the HER2 extracellular domain, and the heavy chain variable region and light chain variable region contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92, 93, 94, 95, 96, or 97 identity with VH shown in SEQ ID NO: 52 and VL shown in SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NOs: 52, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100%, respectively, or SEQ ID NOs: 53, 54, It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68, and VL shown in Sequence ID No. 81, respectively. The amino acid sequence contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NOs. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, and VL shown in SEQ ID NO. 92, respectively.

[0161] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to D3 of the HER2 extracellular domain contains 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 with 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 NOs. 81 or 92. Preferably, the antibody or antigen-binding fragment thereof contains 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 with the VH shown in SEQ ID NOs. 73, 78, 79, or 80, and the VL shown in SEQ ID NOs. 92. More preferably, the antibody or its antigen-binding fragment contains 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 with VH shown in SEQ ID NO: 80 and VL shown in SEQ ID NO: 92, respectively.

[0162] In some specific embodiments, the antibody or antigen-binding fragment of the present invention can specifically bind to D1 of the HER2 extracellular domain, and to at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less) of the amino acid sequence shown in SEQ ID NOs: 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. It includes a heavy chain variable region VH having 99% or 100% identity with the amino acid sequence shown in SEQ ID NOs: 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, with at least 80% identity (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%).

[0163] In one specific embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, and is capable of specifically binding to D1 of the HER2 extracellular domain, and the antibody or antigen-binding fragment is VH as shown in SEQ ID NO: 143 and SEQ ID NOs: 171, 172, 173, 174, 175, 17 Contains 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 with the VL shown in 6, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188, respectively, or contains 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 with the VL shown in SEQ ID NOs. 144, 145, 146, 147, 148, 14 For VH shown in 9, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 or 170 and VL shown in Sequence ID No. 171, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% respectively. It contains an amino acid sequence that is identical to, or contains an amino acid sequence that is identical to, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to, the VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively.

[0164] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to D1 of the HER2 extracellular domain contains 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 with VH shown in any one of SEQ ID NOs. 143, 148, 156, 158, 160, 161, 165, and 169, and VL shown in 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 its antigen-binding fragment contains 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 with VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. More preferably, the antibody or its antigen-binding fragment contains 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 with VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NO: 189, 193, 196, or 198, respectively. Even more preferably, the antibody or its antigen-binding fragment of the present invention contains VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NO: 193.

[0165] The anti-HER2 humanized antibody or its antigen-binding fragment of the present invention has strong binding activity to HER2-expressing cells, does not affect the HER2-mediated signaling pathway, does not cross-react with other members of the human ErbB / HER family (including EGFR, HER3, and HER4), and possesses a high degree of humanization and stability.

[0166] 2. Anti-HER2 biparatopic antibody In one embodiment, the present invention provides an anti-HER2 biparatopic antibody comprising two antigen-binding domains capable of non-competitively and simultaneously binding to the HER2 extracellular domain, wherein the first and second antigen-binding domains each specifically bind to different epitopes on the HER2 extracellular domain. Furthermore, the biparatopic antibody can simultaneously bind to any two epitopes on D1, D3, and D4 of the HER2 extracellular domain. Preferably, the biparatopic antibody can simultaneously bind to D1 and D3 on the HER2 extracellular domain; for example, the first antigen-binding domain specifically binds to D3 of the HER2 extracellular domain, and the second antigen-binding domain specifically binds to D1 of the HER2 extracellular domain. The binding epitopes of the first and second antigen-binding domains are different from the binding epitopes of trastuzumab or pertuzumab.

[0167] The anti-HER2 biparatopic antibody of the present invention has the following functional characteristics:

[0168] (i) Anti-HER2 biparatopic antibodies are tetravalent molecules that can simultaneously bind to two different epitopes on the extracellular domain of HER2, thereby crosslinking HER2 on the cell surface to form antigen-antibody crosslinked multimers or clusters. Clearly, the efficiency and size of such multimer or cluster formation depend on the density of the target antigen on the cell surface, and cluster formation induces endocytosis by the cells, with the rate and intensity of endocytosis positively correlated with the size of the cluster. Specifically, compared to the corresponding anti-HER2 monospecific antibody or its antigen-binding fragment, as well as trastuzumab, biparatopic antibodies exhibit significantly enhanced internalization and have endocytosis rates of over 80% in HER2-overexpressing tumor cells and over 60% in HER2-low-expressing tumor cells.

[0169] (ii) Unlike the corresponding anti-HER2 monospecific antibodies or their antigen-binding fragments and trastuzumab, whose intracellular transport pathway after internalization is primarily recycling to the cell surface, biparatopic antibodies form clusters with HER2 on the cell surface, are internalized, and subsequently, lysosomal transport is their primary intracellular transport pathway. Therefore, the clusters formed by biparatopic antibodies and HER2 can be efficiently transported to lysosomes for degradation.

[0170] (iii) Biparatopic antibodies can effectively induce downregulation of HER2 on the surface of tumor cells, thereby significantly inhibiting the proliferation of HER2-overexpressing tumor cells.

[0171] (iv) Biparatopic antibodies do not interfere with the regulation of HER2 and the downstream signaling pathways mediated by it, including not inducing, blocking, or inhibiting ligand (e.g., NRG-1)-induced HER2 dimerization and the activation of its downstream signaling pathways. Therefore, anti-HER2 biparatopic antibodies do not affect the normal biological function and regulation of HER2 and the downstream signaling pathways mediated by it in normal tissues or cells (e.g., cardiomyocytes).

[0172] (v) Anti-HER2 biparatopic antibodies exhibit high stability, high production yield, and high monomer content (approximately 98% or more).

[0173] Biparatopic antibody formats include bispecificity formats based on scFv or diabody (e.g., scFv-scFv, scFv-Fab, or Fab-scFv), IgG-scFv fusion protein, DVD-Ig, Quadroma, Knob-into-hole, Common Light Chain, CrossMab, CrossFab, SEEDbody, Leucine Zipper, Duobody, IgG1 / IgG2, Dual-acting Fab(DAF)-IgG, and Mab 2This includes, but is not limited to, bispecificity formats (see, for example, Klein et al., mAbs2012, 4:653-663 and the references cited therein). The exemplary anti-HER2 biparatopic antibodies of the present invention employ a DVD-Ig format that exhibits not only high binding activity to HER2-expressing tumor cells but also excellent intracellular integration. Furthermore, the stability of the biparatopic antibodies of the present invention (e.g., high monomer content, approximately 98% or more) can be ensured.

[0174] Anti-HER2 biparatopic antibodies exhibit strong binding activity to HER2-expressing cells and do not cross-react to other members of the human ErbB / HER family (including EGFR, HER3, and HER4).

[0175] Anti-HER2 biparatopic antibodies demonstrate significantly enhanced internalization in HER2-expressing tumor cells. In some embodiments, biparatopic antibodies, compared to corresponding anti-HER2 monospecific antibodies or their antigen-binding fragments and trastuzumab, are effective in HER2-overexpressing tumor cells (IHC3+ or IHC2+ / FISH). + In some embodiments, the biparatopic antibody shows an internalization rate of over 80% in HER2-low-expressing tumor cells (IHC2+ / FISH) compared to the corresponding anti-HER2 monospecific antibody or its antigen-binding fragment and trastuzumab. - It shows an internalization rate of up to 60% in , or IHC1+.

[0176] Anti-HER2 biparatopic antibodies effectively promote HER2 degradation in tumor cells. In some embodiments, Western blot analysis demonstrates that biparatopic antibodies induce HER2 degradation in HER2-overexpressing tumor cells (e.g., BT474 cells), while the corresponding anti-HER2 monospecific antibody or its antigen-binding fragment and trastuzumab fail to induce HER2 degradation in tumor cells. Furthermore, biparatopic antibodies significantly reduce HER2 expression levels on the surface of tumor cells, thereby potently inhibiting the proliferation of HER2-overexpressing tumor cells. In some embodiments, anti-HER2 biparatopic antibodies show a remarkable inhibitory effect on the proliferation of HER2-overexpressing tumor cells (e.g., BT474 cells), with potency comparable to trastuzumab, while the corresponding anti-HER2 monospecific antibody or its antigen-binding fragment fails to inhibit tumor cell proliferation.

[0177] Anti-HER2 biparatopic antibodies maintain the characteristics of the corresponding anti-HER2 monospecific antibody or its antigen-binding fragment, which do not affect the HER2-mediated signaling pathway or interfere with the biological function of HER2, and therefore do not adversely affect the normal biological function of HER2 in normal tissues or cells (e.g., cardiomyocytes). In some embodiments, anti-HER2 biparatopic antibodies do not induce, block, or inhibit ligand (e.g., NRG-1)-induced HER2 dimerization and AKT phosphorylation and / or dephosphorylation in downstream signaling pathways. Therefore, unlike existing HER2-targeted therapies (e.g., trastuzumab- and / or pertuzumab-based therapies), anti-HER2 biparatopic antibodies have a minimal risk of inducing cardiotoxic side effects.

[0178] In another embodiment, the first and second antigen-binding domains of the anti-HER2 biparatopic antibody of the present invention may be derived from any two anti-HER2 monospecific antibodies or antigen-binding fragments thereof of the present invention that do not compete with each other for HER2 binding, or from currently known anti-HER2 antibodies or antigen-binding fragments thereof.

[0179] The exemplary anti-HER2 biparatopic antibody of the present invention can be constructed based on an anti-HER2 antibody or its antigen-binding fragment that specifically binds to D3 of the HER2 extracellular domain as shown in Table 2, and an anti-HER2 antibody or its antigen-binding fragment that specifically binds to D1 of the HER2 extracellular domain as shown in Table 3. The exemplary anti-HER2 biparatopic antibody of the present invention comprises two distinct antigen-binding domains: the first antigen-binding domain comprises at least one CDR that specifically binds to D3 of the HER2 extracellular domain and / or any variable region of the anti-HER2 antibody or its antigen-binding fragment, as shown in Table 2; and the second antigen-binding domain comprises at least one CDR that specifically binds to D1 of the HER2 extracellular domain and / or any variable region of the anti-HER2 antibody or its antigen-binding fragment, as shown in Table 3.

[0180] In some embodiments, the first or second antigen-binding domain of the anti-HER2 biparatopic antibody comprises a heavy chain variable region CDR and / or a light chain variable region CDR, wherein the heavy chain variable region CDR of the first antigen-binding domain comprises the amino acid sequences of one, two, or three CDR regions selected from the heavy chain variable region CDRs listed in Table 2, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, and 9% of HCDR1, HCDR2, and HCDR3, respectively. The amino acid sequences have 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and the light chain variable region CDR of the first antigen-binding domain has amino acid sequences from any one, two, or three CDR regions selected from the light chain variable region CDRs listed in Table 2, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, and 96% identity to LCDR1, LCDR2, and LCDR3, respectively. The amino acid sequences have 97%, 98%, 99%, or 100% identity, and the heavy chain variable region CDR of the second antigen-binding domain has amino acid sequences from any one, two, or three CDR regions selected from the heavy chain variable region CDRs listed in Table 3, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to HCDR1, HCDR2, and HCDR3, respectively. The second antigen-binding domain's light chain variable region CDR contains an amino acid sequence having % identity, and the CDR contains an amino acid sequence of any one, two, or three CDR regions selected from the light chain variable region CDRs 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 with LCDR1, LCDR2, and LCDR3, respectively.

[0181] In some specific embodiments, the first antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D3 of the HER2 extracellular domain and contains 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 with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0182] In some specific embodiments, the first antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D3 of the HER2 extracellular domain and contains 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 with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0183] In some specific embodiments, the second antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D1 of the HER2 extracellular domain and contains 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 with the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0184] In some specific embodiments, the second antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D1 of the HER2 extracellular domain and contains 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 with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0185] In some embodiments, the first or second antigen-binding domain of the anti-HER2 biparatopic antibody further comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3 and / or a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein the heavy chain variable region of the first antigen-binding domain comprises 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, and the light chain variable region of the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, or 91% identity thereto, wherein the light chain variable region of the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, or 91% identity thereto, wherein the light chain variable region of the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, or 91% identity thereto The amino acid sequence contains %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and the heavy chain variable region of the second antigen-binding domain contains any one of the VH amino acid sequences listed in Table 3, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity thereto. The second antigen-binding domain contains an amino acid sequence having 99% or 100% identity, and the light chain variable region of the second antigen-binding domain contains 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 to any one of the VLs listed in Table 3.

[0186] In some specific embodiments, the first antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D3 of the HER2 extracellular domain and includes 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 with the VH and VL, respectively.

[0187] In a specific embodiment, the first antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D3 of the extracellular domain of HER2 and includes 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 with the VH and VL, respectively.

[0188] In some specific embodiments, the second antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D1 of the HER2 extracellular domain and contains 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 with the VH and VL shown in SEQ ID NOs: 169, 190, 191, 192, 193, 194, 195, 196, 197, or 198.

[0189] In a specific embodiment, the second antigen-binding domain of the anti-HER2 biparatopic antibody specifically binds to D1 of the extracellular domain of HER2 and contains 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 with the VH and VL sequences, respectively.

[0190] In some specific embodiments, the anti-HER2 biparatopic antibody comprises a first and a second antigen-binding domain, the first antigen-binding domain comprising the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to said CDRs: HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NOs: 21, 23, 24, 27, 28, 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, or The second antigen-binding domain, shown in SEQ ID NO: 49, is 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 with respect to the following heavy chain variable region CDRs and light chain variable region CDRs: HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 102, HCDR3 shown in SEQ ID NO: 110, LCDR1 shown in SEQ ID NO: 115, LCDR2 shown in SEQ ID NOs: 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and LCDR3 shown in SEQ ID NO: 142.

[0191] In some specific embodiments, the anti-HER2 biparatopic antibody comprises a first and a second antigen-binding domain, the first antigen-binding domain comprising the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to said CDRs: HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NOs: 24, 29, 30, or 31, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 42, sequence number LCDR2 shown in Sequence ID 46 and LCDR3 shown in Sequence ID 49; The second antigen-binding domain includes the following heavy chain variable region CDR and light chain variable region CDR, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to said CDR: HCDR1 shown in Sequence ID 98, HCDR2 shown in Sequence ID 102, HCDR3 shown in Sequence ID 110, LCDR1 shown in Sequence ID 115, LCDR2 shown in Sequence ID 133, and LCDR3 shown in Sequence ID 142.

[0192] In some specific embodiments, the anti-HER2 biparatopic antibody comprises a first and a second antigen-binding domain. The first antigen-binding domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL contain amino acid sequences that are identical to VH shown in SEQ ID NOs. 70, 72, 73, 76, 77, 78, 79, or 80 and VL shown in SEQ ID NOs. 92, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to VH and VL, respectively. The second antigen-binding domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL contain amino acid sequences that are identical to VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NO: 193, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to VH and VL, respectively.

[0193] In some specific embodiments, the anti-HER2 biparatopic antibody comprises a first and a second antigen-binding domain. The first antigen-binding domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL include amino acid sequences that are identical to VH shown in SEQ ID NOs. 73, 78, 79, or 80 and VL shown in SEQ ID NOs. 92, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to VH and VL, respectively. The second antigen-binding domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL contain amino acid sequences that are identical to VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NO: 193, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to VH and VL, respectively.

[0194] The exemplary anti-HER2 biparatopic antibodies of the present invention may also be constructed based on an anti-HER2 antibody or its antigen-binding fragment that specifically binds to domains D1 and D4 of the HER2 extracellular domain as described herein. The exemplary anti-HER2 biparatopic antibodies of the present invention comprise first and second antigen-binding domains, which can bind to HER2 non-competitively and simultaneously. The first antigen-binding domain comprises VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 8, or the first antigen-binding domain comprises VH as shown in SEQ ID NO: 9 and VL as shown in SEQ ID NO: 10, or the first antigen-binding domain comprises at least one of the CDR and / or any one variable region of an anti-HER2 antibody or its antigen-binding fragment as shown in Table 3, and can specifically bind to D1 of the HER2 extracellular domain. The second antigen-binding domain comprises VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO: 6, and can specifically bind to D4 of the HER2 extracellular domain.

[0195] In some embodiments, the first or second antigen-binding domain of the anti-HER2 biparatopic antibody comprises a heavy chain variable region CDR and / or a light chain variable region CDR. The heavy chain variable region CDR of the first antigen-binding domain comprises one, two, or three CDRs from VH shown in SEQ ID NO: 7, or the amino acid sequences shown in SEQ ID NOs: 205, 206, and 207, or variants thereof, wherein the variants comprise a humanized antibody or any other variant. The light chain variable region CDR of the first antigen-binding domain comprises one, two, or three CDRs from VL shown in SEQ ID NO: 8, or the amino acid sequences shown in SEQ ID NOs: 208, 209, and 210, or variants thereof, wherein the variants comprise a humanized antibody or any other variant. Alternatively, the heavy chain variable region CDR of the first antigen-binding domain comprises one, two, or three CDRs from VH shown in SEQ ID NO: 9, or the amino acid sequences shown in SEQ ID NOs: 211, 212, and 213, or their variants, wherein the variants comprise a humanized antibody or any other variant; and the light chain variable region CDR of the first antigen-binding domain comprises one, two, or three CDRs from VL shown in SEQ ID NO: 10, or the amino acid sequences shown in SEQ ID NOs: 214, 215, and 216, or their variants, wherein the variants comprise a humanized antibody or any other variant. Alternatively, the heavy chain variable region CDR of the first antigen-binding domain includes an amino acid sequence of any one, two, or three CDR regions 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, and the light chain variable region CDR of the first antigen-binding domain includes an amino acid sequence of any one, two, or three CDR regions 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 heavy chain variable region (CDR) of the second antigen-binding domain comprises one, two, or three CDRs from VH shown in SEQ ID NO: 5, or the amino acid sequences shown in SEQ ID NOs: 199, 200, and 201, or their variants, wherein the variants include humanized antibodies or any other variants. The light chain variable region (CDR) of the first antigen-binding domain comprises one, two, or three CDRs from VL shown in SEQ ID NO: 6, or the amino acid sequences shown in SEQ ID NOs: 202, 203, and 204, or their variants, wherein the variants include humanized antibodies or any other variants.

[0196] The exemplary anti-HER2 biparatopic antibodies of the present invention may also be constructed based on an anti-HER2 antibody or its antigen-binding fragment that specifically binds to domains D3 and D4 of the HER2 extracellular domain as described herein. The exemplary anti-HER2 biparatopic antibodies of the present invention comprise first and second antigen-binding domains, the first and second antigen-binding domains which can bind to HER2 non-competitively and simultaneously, the first antigen-binding domain comprising at least one CDR and / or any one variable region or antigen-binding fragment of the anti-HER2 antibody that specifically binds to D3 of the HER2 extracellular domain as shown in Table 2, and the second antigen-binding domain comprising VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO: 6, which can specifically bind to D4 of the HER2 extracellular domain.

[0197] In some embodiments, the first or second antigen-binding domain of the anti-HER2 biparatopic antibody includes a heavy chain variable region CDR and / or a light chain variable region CDR. The heavy chain variable region CDR of the first antigen-binding domain includes an amino acid sequence of any one, two, or three CDR regions 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, and the light chain variable region CDR of the first antigen-binding domain includes an amino acid sequence of any one, two, or three CDR regions listed in Table 2, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, or 93% identity thereto. The amino acid sequence comprises an amino acid sequence having 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and the heavy chain variable region CDR of the second antigen-binding domain comprises one, two, or three CDRs from VH shown in SEQ ID NO: 5, or the amino acid sequence shown in SEQ ID NOs: 199, 200, and 201, or a variant thereof, the variant comprising a humanized antibody or any other variant; and the light chain variable region CDR of the first antigen-binding domain comprises one, two, or three CDRs from VL shown in SEQ ID NO: 6, or the amino acid sequence shown in SEQ ID NOs: 202, 203, and 204, or a variant thereof, the variant comprising a humanized antibody or any other variant.

[0198] In another embodiment, an exemplary anti-HER2 biparatopic antibody of the present invention can be constructed in DVD-Ig format (see U.S. Patent No. 7612181). The first or second antigen-binding domain of the anti-HER2 biparatopic antibody may be either Fv form / domain or Fab / IgG form / domain, i.e., if the first antigen-binding domain is Fv, the second antigen-binding domain is Fab or IgG, and if the second antigen-binding domain is Fv, the first antigen-binding domain is Fab or IgG. The Fv domain is fused or operably linked to the Fab or IgG domain via a linker, which not only has low immunogenicity but also ensures the stability of the biparatopic antibody, and a flexible peptide is preferably used as the linker.

[0199] As used herein, the term “linker” refers to a portion that links two compounds, for example, two polypeptide molecules (including, but not limited to, unmodified or modified amino acids or amino acid sequences). A linker may consist of one or more linking units, or may include a linking unit and at least one spacer designed to separate the linking unit and the compound by a specific distance.

[0200] The term "operably linked" refers to the linking of amino acid sequences, peptides, or proteins having different functional characteristics, such as the linking of an Fv domain to a Fab or IgG domain via a linker as described herein.

[0201] In some embodiments, the first antigen-binding domain of the anti-HER2 biparatopic antibody is an Fv domain that specifically binds to D3 of the HER2 extracellular domain and contains the heavy chain variable region CDR and light chain variable region CDR of the anti-HER2 antibody or its antigen-binding fragment shown in Table 2, and / or the heavy chain variable region VH and light chain variable region VL. The second antigen-binding domain of the anti-HER2 biparatopic antibody is a Fab domain or IgG domain that specifically binds to D1 of the HER2 extracellular domain and contains the heavy chain variable region CDR and light chain variable region CDR of the anti-HER2 antibody or its antigen-binding fragment shown in Table 3, and / or the heavy chain variable region VH and light chain variable region VL. The C-terminus of the VH domain in the Fv domain is fused 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 or operably linked to the N-terminus of the VL domain in the Fab or IgG domain via a linker sequence.

[0202] In some specific embodiments, the linker contains GGGGS sequences (G4S) of different copy numbers, for example, 1, 2, 3, 4, or 5 copies. The number of copies of the G4S linking the VH domain in the Fv domain of the anti-HER2 biparatopic antibody to the VH domain in the Fab or IgG domain of the biparatopic antibody may be the same as or different from the number of copies of the G4S linking the VL domain in the Fv domain of the biparatopic antibody to the VL domain in the Fab or IgG domain of the biparatopic antibody. Preferably, the number of copies of the G4S linking the VH domain between the Fv domain and the Fab or IgG domain is different from the number of copies of the G4S linking the VL domain between the two, with the number of linker copies linking the two VH domains being preferably 1 and the number of linker copies linking the two VL domains being preferably 3, thereby effectively ensuring the structural stability of the anti-HER2 biparatopic antibody of the present invention and enhancing internalization in HER2-expressing tumor cells.

[0203] In specific embodiments, the anti-HER2 biparatopic antibody comprises a first and a second antigen-binding domain, the first antigen-binding domain comprising the VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 2, and the second antigen-binding domain comprising the VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 3. The first antigen-binding domain is an Fv domain, and the second antigen-binding domain is a Fab or IgG domain. The C-terminus of the VH domain in the Fv domain is a linker (e.g., (G4S)). n (wherein n is an integer greater than 0, e.g., 1 to 3), preferably via (G4S)1) the N-terminus of the VH domain in the Fab or IgG domain. The C-terminus of the VL domain in the Fv domain is linked by a linker (e.g., (G4S) n (wherein n is an integer from 1 to 3), preferably ligated to the N-terminus of the VL domain in the Fab or IgG domain via (G4S)3).

[0204] In another embodiment, the anti-HER2 biparatopic antibody of the present invention may further comprise a constant region including a heavy chain constant region and a light chain constant region of the antibody. The heavy chain constant region of the present invention comprises native and mutant forms of the Fc region of the human IgG heavy chain constant region, and also comprises a cleaved polypeptide form containing a hinge region that promotes dimerization. In some embodiments, the Fc region comprises the CH2 and CH3 domains of the antibody. Fusion proteins containing the Fc portion (and oligomers formed therefrom) offer the advantages of easy purification by protein A or protein G affinity chromatography and an extended serum half-life. Preferably, the Fc region is derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. Here, the position of specific amino acid residues in the Fc region is determined according to the EU numbering system.

[0205] One function of the Fc region of an antibody is to generate "effector functions" by the immune system, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC), when the antibody binds to its target. 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), and immune cells include 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 the binding of Fc to complement system proteins, such as C1q (Ward et al., Ther Immunol 1995, 2:77-94).

[0206] In some embodiments, the anti-HER2 biparatopic antibody of the present invention includes an engineered IgG Fc region for suppressing Fc-mediated effector function. Exemplary antibodies with reduced effector function include an Fc region containing the following amino acid mutations. N297A or N297Q (IgG1) S267E / L328F(IgG1) L234A / L235A (IgG1) L234F / L235E / P331S(IgG1) C220S / C226S / C229S / P238S(IgG1) C226S / C229S / E233P / L234V / L235A(IgG1) V234A / G237A(IgG2) H268Q / V309L / A330S / A331S(IgG2) L235A / G237A / E318A(IgG4)

[0207] Preferably, the engineering Fc region is a human IgG1 Fc having the amino acid substitution L234F / L235E / P331S (EU numbering system), which can reduce the binding of the Fc region to one or more FcγR and C1q (Oganesyan et al., Acta Crystallogr D Biol Crystallogr 2008, 64:700-704, US5624821, US6194551). The FcγR family includes FcγRI (also known as CD64), such as isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (also known as CD32), such as isoforms FcγRIIa, FcγRIIb, and FcγRIIc; and FcγRIII (also known as CD16), such as isoforms FcγRIIIa and FcγRIIIb (Jefferis et al., Immunol Lett 2002, 82:57-65). Among the FcγRs, FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa can induce ADCC, endocytosis, phagocytosis, and / or cytokine release. Binding properties include binding specificity and binding affinity constant (K). D ), as well as the dissociation rate and binding rate (k each) dis and k a This includes, but is not limited to, the following. Those skilled in the art can analyze whether the engineering Fc region has altered ADCC and / or CDC activity based on one or more of these binding properties. In some embodiments, the heavy chain constant region and light chain constant region in the anti-HER2 biparatopic antibody of the present invention may be derived from the heavy chain constant region of human IgG1 or IgG4 (either wild-type or mutant, e.g., the wild-type human IgG1 constant region shown in SEQ ID NO: 231), and the human kappa (κ) or lambda (λ) constant region.

[0208] In some specific embodiments, the heavy chain constant region of an anti-HER2 biparatopic antibody containing human IgG1 Fc having the amino acid substitution L234F / L235E / P331S (an amino acid substitution according to EU numbering, the sequence of which is shown in SEQ ID NO: 232) exhibits reduced binding affinity to one or more FcγRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIa) and C1q. In some embodiments, introducing the L234F / L235E / P331S mutation into the heavy chain constant region of an anti-HER2 biparatopic antibody results in loss of binding to FcγRI, FcγRIIa(167H), FcγRIIb, FcγRIIIa(176V), and / or FcγRIIIa(176F). In some embodiments, introducing the L234F / L235E / P331S mutation into the heavy chain constant region of an anti-HER2 biparatopic antibody results in loss of binding to C1q. In some embodiments, introducing the L234F / L235E / P331S mutation into the heavy chain constant region of an anti-HER2 biparatopic antibody does not affect the binding affinity of the biparatopic antibody to FcRn. In some embodiments, introducing the L234F / L235E / P331S mutation into the heavy chain constant region of an anti-HER2 biparatopic antibody can significantly reduce the ADCC activity of the biparatopic antibody. In one embodiment, the biparatopic antibody has significantly attenuated or undetectable ADCC activity compared to an antibody without the amino acid mutation in its Fc region. Such attenuation or elimination of ADCC activity is likely caused by a significant decrease in the binding affinity of the biparatopic antibody to FcγR.

[0209] In some specific embodiments, the anti-HER2 biparatopic antibody comprises a light chain constant region, which is selected from a kappa constant region (as shown in SEQ ID NO: 233).

[0210] In one aspect, the anti-HER2 bispecific antibody of the present invention comprises four polypeptide chains, two of the polypeptide chains comprising VH1-L1-VH2-C-(Fc)n, where VH1 represents the heavy chain variable region of a first antigen-binding domain that specifically binds to D3 of the HER2 extracellular domain, L1 represents a linker, VH2 represents the heavy chain variable region of a second antigen-binding domain that specifically binds to D1 of the HER2 extracellular domain, C represents the heavy chain constant region CH1, Fc represents the heavy chain constant region Fc domain, n is 0 or 1, and the other two polypeptide chains comprise VL1-L2-VL2-CL, where VL1 represents the light chain variable region of a first antigen-binding domain that specifically binds to D3 of the HER2 extracellular domain, L2 represents a linker, VL2 represents the light chain variable region of a second antigen-binding domain that specifically binds to D1 of the HER2 extracellular domain, and CL is an IgG light chain constant region.

[0211] In some embodiments, VH1 comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 11, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 24, 29, 30 or 31, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 37, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, respectively, to said HCDR1, HCDR2 and HCDR3, L1 comprises a linker having the amino acid sequence shown in SEQ ID NO: 229, VH2 comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 98, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 102, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 110, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, respectively, to said HCDR1, HCDR2 and HCDR3, and Fc comprises an engineered Fc domain comprising a human IgG1 Fc having the amino acid substitution L234F / L235E / P331S (EU numbering system) having the amino acid sequence shown in SEQ ID NO: 232.

[0212] In some specific embodiments, VH1 comprises a VH having an amino acid sequence shown 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, and VH2 comprises a VH having an amino acid sequence shown 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.

[0213] In some embodiments, VL1 comprises LCDR1 having an amino acid sequence shown in SEQ ID NO: 42, LCDR2 having an amino acid sequence shown in SEQ ID NO: 46, and LCDR3 having an amino acid sequence shown in SEQ ID NO: 49, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said LCDR1, LCDR2 and LCDR3 respectively, L2 comprises a linker having an amino acid sequence shown in SEQ ID NO: 230, VL2 comprises LCDR1 having an amino acid sequence shown in SEQ ID NO: 115, LCDR2 having an amino acid sequence shown in SEQ ID NO: 133, and LCDR3 having an amino acid sequence shown in SEQ ID NO: 142, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said LCDR1, LCDR2 and LCDR3 respectively, and CL is selected from a human κ constant region or a human λ constant region, preferably a human κ constant region (amino acid sequence shown in SEQ ID NO: 233).

[0214] In some specific embodiments, VL1 includes a VL 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 to the amino acid sequence shown in SEQ ID NO: 193, or a VL having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 193.

[0215] The anti-HER2 biparatopic antibody of the present invention can specifically and simultaneously bind to two different epitopes on the extracellular domain of HER2, effectively crosslinking HER2 on the cell surface to form antigen-antibody crosslinked multimers or clusters, thereby inducing endocytosis and facilitating the transport of internalized clusters to lysosomes for degradation. As a result, the biparatopic antibody can significantly downregulate HER2 expression on the cell surface, reduce HER2 dimerization, and thus inhibit the proliferation of HER2-overexpressing tumor cells. Furthermore, the anti-HER2 biparatopic antibody of the present invention does not interfere with the HER2-mediated signaling pathway and therefore does not interfere with the normal biological function of HER2 in normal tissues / cells.

[0216] 3. Anti-HER2 biparatopic antibody-drug conjugate (ADC) The present invention further provides an anti-HER2 biparatopic ADC comprising a small molecule toxin compound conjugated to the anti-HER2 biparatopic antibody of the present invention via a cleavable linker, having one or more of the following functional characteristics.

[0217] (i) Anti-HER2 biparatopic ADCs are tetravalent molecules that specifically bind to two non-overlapping epitopes of HER2 on the surface of tumor cells, and thus effectively crosslink HER2, forming crosslinked multimers or clusters on the tumor cell membrane. In one embodiment, cluster formation promotes rapid and efficient endocytosis of the ADC by the cell, and furthermore, the intracellular transport pathway after endocytosis changes from recycling to lysosomal transport, significantly increasing the efficiency of ADC transport to lysosomes. After degradation in lysosomes, more small molecule toxic compounds can be released into the cytoplasm. Thus, biparatopic ADCs exhibit stronger targeted cytotoxicity compared to existing HER2-targeted therapies (e.g., trastuzumab, pertuzumab, T-DM1, DS-8201) in HER2-overexpressing tumor cells (IHC3+ or IHC2+ / FISH). + ) showed excellent killing activity in HER2-low-expressing tumor cells (IHC2+ / FISH). - In another aspect, the released small molecule toxin compounds are hydrophobic, permeable to cell membranes, and passively diffuse into the tumor microenvironment, exerting a bystander effect, i.e., enabling the killing of adjacent low-HER2 or non-HER2-expressing tumor cells. In summary, compared to conventional ADCs, biparatopic ADCs demonstrate a broader range of tumor cell-killing activity and a reduced likelihood of developing drug resistance.

[0218] (ii) Anti-HER2 biparatopic ADCs exhibit cytotoxic activity in tumors that have acquired resistance to or relapsed treatment with trastuzumab, pertuzumab, T-DM1, or DS-8201. As a result, they may address the drug resistance issues associated with existing HER2-targeted therapies.

[0219] (iii) Anti-HER2 biparatopic ADCs do not interfere with HER2 dimerization and the modulation of HER2-mediated downstream signaling pathways. As a result, they do not interfere with the normal biological function of HER2 in cardiomyocytes and therefore significantly reduce the risk of cardiotoxic side effects.

[0220] (iv) Anti-HER2 biparatopic ADCs exhibit significantly reduced Fc receptor binding affinity, thus posing a very low safety risk related to Fc receptors.

[0221] The ADCs described herein can be expressed by formula (I): Ab-(LD)p(I) In the formula, Ab represents the anti-HER2 biparatopic antibody of the present invention, D stands for small molecule toxin compound (Drug), L represents a cleavable linker that connects Ab to D. p represents the copy number of the (LD) bonded with Ab, and is in the range of 2 to 8.

[0222] Ab of the ADC described herein is an anti-HER2 biparatopic antibody of the present invention comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 2, specifically binds to D3 of the HER2 extracellular region, and is an Fv domain, and the second antigen-binding domain comprises VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 3, specifically binds to D1 of the HER2 extracellular region, and is a Fab domain or an IgG domain.

[0223] In some embodiments, the first antigen-binding domain of an anti-HER2 biparatopic ADC comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL contain amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0224] In some embodiments, the second antigen-binding domain of the anti-HER2 biparatopic ADC comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL contain amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0225] In some embodiments, the first antigen-binding domain of the anti-HER2 biparatopic ADC includes 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 to the VH shown in SEQ ID NO: 73, 78, 79, or 80, and / or includes 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 to the VL shown in SEQ ID NO: 92.

[0226] In some embodiments, the second antigen-binding domain of the anti-HER2 biparatopic ADC comprises 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 to the VH sequence shown in SEQ ID NO: 169, and / or comprises 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 to the VL sequence shown in SEQ ID NO: 193.

[0227] The D in ADC is a small molecule toxin compound that, when not bound to an antibody, exhibits cytotoxic or cell proliferation inhibitory effects against both tumor cells and normal cells. However, when a small molecule toxin compound is bound to the anti-HER2 biparatopic antibody of the present invention to form an ADC, the ADC exerts its cytotoxic and / or bystander-killing effects only after it has been internalized, transported to the lysosomes of HER2-expressing target cells, and released from the ADC via enzymatic action, hydrolysis, oxidation, or any other mechanism. Small molecule toxin compounds include cytotoxins and chemotherapeutic agents.

[0228] In some embodiments, the cytotoxins include tubulin inhibitors and DNA alkylating agents. The tubulin inhibitors include eribulin, auristatin derivatives (e.g., MMAE, MMAF, MMAD), tubulysin, cryptomycin, and maytansinoid derivatives (e.g., DM1, DM2, DM3, DM4). The DNA alkylating agents include topoisomerase inhibitors (e.g., camptothecin derivatives such as SN-38, exatecan, Dxd, etc.), pyrrolobenzodiazepine (PBD), calicheamicin and its derivatives (e.g., N-acetylcalicheamicin [CMC]), and duocarmycin. A preferred small molecule toxin compound is eribulin.

[0229] The molecular structure of eribulin is represented by formula (II).

Chemical formula

[0230] The term "eribulin" refers to a synthetic analog of halichondrin B, a macrocyclic compound isolated from the sponge Halichondria okadai. As a microtubule dynamics inhibitor, eribulin binds to tubulin, thereby inhibiting the formation of the mitotic spindle and arresting cell cycle division at the G2 / M phase. Exemplary structures of eribulin or its analogs, as well as their synthetic methods, are described in International Publication No. WO 1999 / 065894, International Publication No. WO 2004 / 034990, Chinese Patent No. ZL201910197071.8, and / or Chinese Patent No. ZL201910509222.9, the disclosures of which are incorporated herein by reference.

[0231] In some embodiments, the small molecule toxin compound is eribulin, which is characterized by a broad clinical therapeutic window and low off-target cytotoxicity.

[0232] In some embodiments, the chemotherapeutic agent may be a natural or synthetic compound, including, but not limited to, alkylating chemotherapeutic agents and other compounds having alkylating activity (e.g., nitrogen mustard, ethyleneimine compounds, alkyl sulfonates, nitrosourea, cisplatin, dacarbazine), antimetabolites (e.g., folate antagonists, purine antagonists, or pyrimidine antagonists), mitotic inhibitors (e.g., vinca alkaloids and podophyllotoxin derivatives), and cytotoxic antibiotics (e.g., anthracycline antibiotics including daunorubicin and doxorubicin, as well as actinomycin and bleomycin).

[0233] The cleavable linker is stable outside HER2-expressing tumor cells, allowing the ADC of the present invention to maintain structural stability in vitro or in the bloodstream without causing systemic toxicity through the untargeted or random release of small molecule toxic compounds (off-target effects). On the other hand, after endocytosis to HER2-expressing tumor cells and transport to lysosomes, the linker is rapidly cleaved, releasing the small molecule toxic compound to kill the tumor cells. A cleavable linker refers to any linker containing a cleavable moiety. As used herein, the term "cleavable moiety" refers to any cleavable chemical bond known in the art, including, but not limited to, acid-unstable bonds, protease / peptidase-unstable bonds, photosensitive bonds, esterase-unstable bonds, and disulfide bonds. A linker containing a cleavable moiety allows the small molecule toxic compound to be released from the ADC via cleavage at a specific site within the linker.

[0234] In some embodiments, the cleavable linker comprises a cleavable peptide moiety, which, after being cleaved by an intracellular protease (e.g., endosomal protease, lysosomal protease, or tumor-associated protease), releases a small molecule toxic compound from the ADC to kill tumor cells. In some embodiments, the cleavable peptide moiety 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 unnatural amino acid analogs, such as citrulline (abbreviated as Cit). In some embodiments, the amino acid unit comprises a dipeptide, tripeptide, or tetrapeptide having an amino acid sequence such as Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG), which can be cleaved by cathepsin B, for example. Preferred amino acid units include VC and GGFG.

[0235] In some embodiments, the cleavable linker may include at least one spacer linking a small molecule toxin compound (D) to the anti-HER2 biparatopic antibody (Ab) of the present invention, wherein the spacer includes a spacer linked to the antibody and / or a second spacer linked to the small molecule compound.

[0236] In some embodiments, the spacer linked to the antibody may be hydrophilic to enhance the hydrophilicity of the ADC, thereby improving its stability, reducing aggregation of the ADC product, and decreasing its immunogenicity. An exemplary spacer comprises one or more polyethylene glycols (PEGs), e.g., 1, 2, 3, 4, 5, or 6 PEGs, with 2 PEGs being preferred. In some examples, the spacer is linked to the antibody via maleimide (Mal), where the spacer linked to the antibody via Mal may also be referred to herein as a "Mal-spacer." In some examples, the Mal-spacer comprises one or more PEG moieties (e.g., 2 PEGs).

[0237] In some embodiments, a second spacer linked to a small molecule compound is used to bind the cleavable portion of a cleavable linker (e.g., a cleavable peptide) to the small molecule compound. In some examples, the second spacer linked to the small molecule compound exhibits autodegradability, which facilitates the complete release of the small molecule compound into the target cell, i.e., the small molecule compound released into the target cell has no residual spacer portion or other modifying groups bound to it, ensuring that the antitumor activity of the released small molecule compound is not affected by the presence of modifying groups. In some examples, the autodegradable spacer comprises a p-aminobenzyl unit, where p-aminobenzyl alcohol (PABOH) is bound to the cleavable portion of the cleavable linker (e.g., the amino acid unit of a cleavable peptide) via an amide bond, and a carbamate, methylcarbamate, or 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-degrading spacer contains p-aminobenzylcarbonyl (PAB) as shown in formula (III), and the self-degradation of PAB is accompanied by a spontaneous 1,6-elimination reaction (Jain et al., Pharmaceutical Research 2015, 32:3526-3540). [ka]

[0238] In some embodiments, the cleavable linker comprises a Mal-spacer and a cleavable peptide moiety. In some embodiments, the spacer comprises a PEG moiety (e.g., two PEGs) and the cleavable peptide moiety comprises an amino acid unit (e.g., dipeptide VC or tetrapeptide GGFG). In some embodiments, the cleavable linker comprises a covalently bonded Mal-spacer-amino acid unit, where the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG. In some embodiments, the cleavable linker comprises a covalently bonded Mal-spacer-amino acid unit-PAB, where the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG. In some examples, the spacer is (PEG) m Here, m is an integer from 1 to 5, preferably 2. In some examples, the cleavable linker includes Mal-(PEG)2 and VC. In some examples, the cleavable linker includes Mal-(PEG)2 and GGFG.

[0239] In some embodiments, the cleavable linker comprises a structure:Mal-spacer cleavable peptide moiety. In some examples, the cleavable linker comprises structures:Mal-(PEG)2-VC and Mal-(PEG)2-GGFG.

[0240] In some embodiments, the cleavable linker comprises a Mal-spacer, a cleavable peptide moiety, and a second spacer. In some examples, the spacer comprises a PEG moiety (e.g., two PEGs), the cleavable peptide moiety comprises an amino acid unit (e.g., dipeptide VC), and the second spacer is self-degradable (e.g., PAB). In some examples, the cleavable linker comprises Mal-(PEG)2, VC, and PAB.

[0241] In some embodiments, the cleavable linker comprises the structure: Mal-spacer-cleavable peptide moiety-second spacer. In some examples, the cleavable linker comprises the structure: Mal-(PEG)2-VC-PAB.

[0242] In some embodiments, the Mal-spacer of the cleavable linker is bound to one or more amino acid residues of the antibody component in the ADC of the present invention. For example, the Mal-spacer may be bound to the antibody via a thiol group, including conjugation to the sulfhydryl groups 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 a cysteine ​​residue of the antibody, thereby binding the Mal-spacer to the antibody. In some embodiments, the maleimide group of the Mal-spacer can react with the sulfhydryl group of a cysteine ​​residue at a specific position in the constant region and / or variable region of the antibody. Furthermore, the maleimide group of the Mal-spacer can be bound to free cysteine ​​residues released upon reduction of disulfide bonds and / or interchain disulfide bonds within the antibody hinge region. In some examples, the maleimide group of the Mal-spacer is bound to a cysteine ​​residue within the antibody hinge region. Multiple free cysteine ​​residues can be generated by subjecting the interchain disulfide bonds within the antibody hinge region to chemical reactions (e.g., reduction, pH adjustment, or hydrolysis). Alternatively, engineered cysteine ​​residues can be generated by manipulating one or more amino acid residues at specific sites in the antibody constant region using DNA recombination techniques (e.g., by substituting or inserting cysteine ​​residues).

[0243] In some embodiments, the second spacer of the cleavable linker is bound to a small molecule compound, where the small molecule compound is eribulin or a derivative thereof. In some embodiments, the second spacer binds the cleavable peptide moiety in the cleavable linker to the C-35 amine of eribulin, where the second spacer is self-degrading (e.g., PAB), and the cleavable peptide moiety contains VC. The self-degrading spacer spontaneously degrades after enzymatic cleavage of the cleavable peptide moiety, resulting in the release of naturally active eribulin from the ADC. In some examples, the self-degrading spacer is PAB, which binds the cleavable peptide moiety VC to the C-35 amine of eribulin to construct an ADC having the structure Mal-(PEG)2-VC-PAB-eribulin.

[0244] In some embodiments, the cleavable portion of the cleavable linker (e.g., a cleavable polypeptide) can be directly bound to a small molecule toxic compound component of the ADC, where the small molecule toxic compound is eribulin or a derivative thereof. In some embodiments, the cleavable polypeptide comprises an amino acid unit, the amino acid unit comprising GGFG, and this tetrapeptide is bound to the C-35 amine of eribulin via its carboxyl group, thereby constructing an ADC having the structure Mal-(PEG)2-GGFG-eribulin. The approach of directly binding the GGFG tetrapeptide in the cleavable linker to eribulin also ensures that eribulin can be released in its naturally active form after the cleavable linker of the ADC is cleaved in target cells, thereby allowing eribulin to exert a target tumor cell-killing effect and / or bystander effect.

[0245] In some embodiments, the ADC structure of the present invention comprises the anti-HER2 biparatopic antibody of the present invention conjugated to eribulin or a derivative thereof via a cleavable linker, the cleavable linker comprising a Mal-spacer, a cleavable peptide moiety and / or a second spacer. In some embodiments, the ADC structure comprises the anti-HER2 biparatopic antibody of the present invention conjugated to the C-35 amine of eribulin via a cleavable linker, the cleavable linker comprising Mal-(PEG)2-VC-PAB or Mal-(PEG)2-GGFG. Free cysteine ​​residues released upon reduction of disulfide bonds and / or interchain disulfide bonds within the antibody hinge region are bound to the Mal group of the cleavable linker.

[0246] In some embodiments, the ADC of the present invention comprises the following components. [ka]

[0247] In some embodiments, the ADC of the present invention comprises the anti-HER2 biparatopic antibody of the present invention conjugated to a drug payload, the drug payload comprising Mal-(PEG)2-VC-PAB-eribulin and Mal-(PEG)2-GGFG-eribulin. In some embodiments, the ADC comprises the anti-HER2 biparatopic antibody of the present invention conjugated to a Mal group of the drug payload via a cysteine ​​residue. More specifically, free cysteine ​​residues released upon reduction of disulfide bonds and / or interchain disulfide bonds in the hinge region of the biparatopic antibody are conjugated to a Mal group of the drug payload.

[0248] In some embodiments, the present invention provides an ADC having the following formula. [ka] In the formula, Ab represents the anti-HER2 biparatopic antibody of the present invention, and p is 2 to 8.

[0249] In formula (I), p is also referred herein to as the drug-to-antibody ratio (DAR) or the number of payload molecules, i.e., the number of small molecule compounds complexed with the antibody, where p is in the range of 2 to 8, for example, 4 to 8. The DAR value of the ADC of the present invention, in which the anti-HER2 biparatopic antibody is bound to eribulin via a cleavable linker, exhibits desirable properties, namely, the ADC exhibits not only sufficient killing activity but also sufficient stability. Generally, higher DAR values ​​(e.g., p>8) may result in aggregation and precipitation of the ADC due to the strong hydrophobicity of the small molecule toxin compounds, which can promote hydrophobic interactions in an aqueous environment, potentially even increasing the in vivo safety risk, while lower DAR values ​​(e.g., p<2) may result in reduced killing activity of the ADC against tumor cells, thereby affecting therapeutic efficacy. In some embodiments, the optimal DAR value is about 4 to 8. The average DAR value (i.e., average number of payload molecules or average p) of the anti-HER2 biparatopic ADC of the present invention can be calculated using data obtained from conventional analyses known in the art (e.g., reversed-phase LC-MS mass spectrometry and / or HIC-HPLC).

[0250] 4. Method for preparing the antibody of the present invention 4.1 Polynucleotides, vectors, and host cells In one embodiment, the present invention provides nucleic acids encoding an anti-HER2 antibody or an antigen-binding fragment thereof, or an anti-HER2 biparatopic antibody. The present invention also includes polynucleotide modifiers encoding the amino acid sequences described herein.

[0251] The nucleotide sequences corresponding to the amino acid sequences described in this invention, probes or primers for use in nucleic acid isolation, or database-queriable sequences can be obtained by back-translation of the amino acid sequences.

[0252] Using polymerase chain reaction (PCR) procedures, the anti-HER2 antibody or its antigen-binding fragment, or the DNA sequence encoding the anti-HER2 biparatopic antibody of the present invention, can be isolated and amplified. Oligonucleotides that define the desired ends of the DNA fragment assembly are used as 5' and 3' primers. The oligonucleotides may further contain recognition sites of restriction endonucleases to facilitate the combined insertion of the amplified DNA fragments into an expression vector. PCR techniques are described, for example, in Saiki et al., Science 1988, 239:487-491; Wu et al., (eds), Recombinant DNA Methodology, 1989, Academic Press, pp.189-196; and Innis et al., (eds), PCR Protocols: A Guide to Methods and Applications, 1990, Academic Press.

[0253] The nucleic acids of the present invention include both single-stranded and double-stranded forms of DNA and RNA, as well as their corresponding complementary sequences, and include isolated nucleic acids, preferably obtained in substantially pure form and in sufficient quantity or concentration to identify, manipulate, and recover their component nucleotide sequences, using standard biochemical methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory), and containing DNA or RNA derived from at least one isolation. Preferably, such sequences include those provided and / or constructed in the form of internal untranslated sequences or open reading frames interrupted by introns not commonly present in eukaryotic genes. The sequence of untranslated DNA may be present at the 5' or 3' end of the open reading frame, and this sequence does not interfere with the manipulation or expression of the coding region.

[0254] The anti-HER2 antibody or its antigen-binding fragment or anti-HER2 biparatopic antibody of the present invention can be prepared by the following steps: site-directed mutagenesis of nucleotides in the DNA encoding the anti-HER2 antibody or its antigen-binding fragment or anti-HER2 biparatopic antibody using PCR mutagenesis or other techniques known to those skilled in the art to generate a DNA-coding variant, and then expression of the recombinant DNA in a cell culture as outlined herein. Alternatively, the anti-HER2 antibody or its antigen-binding fragment or anti-HER2 biparatopic antibody can also be prepared by in vitro synthesis using established techniques.

[0255] As is known to those skilled in the art, due to the degeneracy of the gene code, the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 biparatopic antibody of the present invention, can be encoded by a very large number of nucleic acids, each of which falls within the scope of the present invention and can be produced using standard techniques. Therefore, once a specific amino acid sequence is identified, those skilled in the art can prepare a variety of different nucleic acids by simply making one or more codon modifications to the respective coding sequences of the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 biparatopic antibody of the present invention, without altering their amino acid sequences.

[0256] In another embodiment, the present invention also provides an expression vector for nucleic acids encoding an anti-HER2 antibody or its antigen-binding fragment, or an anti-HER2 biparatopic antibody, as described herein.

[0257] The anti-HER2 antibody or its antigen-binding fragment, or the nucleic acid encoding the anti-HER2 biparatopic antibody of the present invention, can be constructed into a vector suitable for introduction into host cells to express the target protein. Typical vector components 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 ligated to the promoter.

[0258] As used herein, the term “operatably linked” refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, so that the regulatory sequence modulates the transcription and / or translation of the other nucleic acid sequence.

[0259] Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements for controlling expression (including promoter sequences, transcription start sequences, enhancer sequences, selectable elements, and reporter genes). Furthermore, vectors may also contain replication origins. Vectors may also contain components that facilitate their entry into cells, including, but are not limited to, viral particles, liposomes, or protein coats.

[0260] In another embodiment, the present invention also provides host cells comprising the anti-HER2 antibody or its antigen-binding fragment, or a nucleic acid or expression vector encoding the anti-HER2 biparatopic antibody.

[0261] The cells include eukaryotic cells, such as, but not limited to, SV40-transformed monkey kidney cell lines CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell lines (293 or subclones of 293 cells in suspension culture, 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), and mouse Sertoli cells (TM4, Mather, Biol). These may include mammalian host cells such as 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 hepatocytes (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human hepatocellular carcinoma cell lines (HepG2, ATCC, HB-8065), mouse mammary tumor cells (MMT060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC5 cells, or FS4 cells.

[0262] 4.2 Screening of the anti-HER2 antibody of the present invention The anti-HER2 antibodies provided by the present invention may be mouse 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.) can be used in the present invention to prepare monoclonal antibodies that specifically bind to HER2.

[0263] In some embodiments, polypeptides of any fragment in the HER2 amino acid sequence (preferably the HER2 extracellular domain) can be used as target antigens for immunizing mice. Antibody variable region genes are amplified from mature B cells of immunized mice, and a phage display library containing mouse antibody variable regions presented on the surface of phages is constructed using phage display technology. Subsequently, the library is panned against a specific target molecule (e.g., a target antigen used to immunize mice) to detect the interaction between the antibody variable region presented on the phage surface and the target antigen. The antibody variable region library is screened and amplified by an in vitro selection method similar to natural selection.

[0264] In some embodiments, the present invention includes constructing a phage display library and performing panning against a target antigen to screen for antibodies that recognize the target antigen from an antibody library. Antibodies of the present invention can be produced using various phage display methods known in the art. For example, U.S. Patent No. 5,223409, U.S. Patent No. 5,622699, and U.S. Patent No. 6,068829 disclose methods for preparing phage libraries. Alternatively, a phage display library can be constructed according to the method described in "Antibody Phage Display: Methods and Protocols (edited by O'Brien and Aitken)". In some embodiments, nucleic acids encoding the antibody variable region can be inserted into the phage coat protein gene to enable the phage to present the antibody variable region on its surface. Simultaneously, the phage contains the nucleic acid encoding the antibody variable region, thereby achieving a link between the phenotype and genotype of the antibody variable region.

[0265] Regarding antibody (or single-chain antibody) screening by phage display, the VH and / or VL regions of antibodies (or single-chain antibodies) from a considerable library can be expressed on the surface of fibrous phage particles, thereby allowing them to be paired to form binding domains. The phages can be selected from the library based on their recognition and binding to target antigens via the binding domains they present.

[0266] In some embodiments, panning can be achieved by infecting a host bacterium with a phage, which is then grown and amplified within the host. The phages, secreted by the host bacterium and displaying single-chain antibody fragments on their surface, are collected and subjected to multiple panning cycles as needed until phages capable of selectively or specifically binding to a target antigen are obtained. Finally, the amino acid sequence of the antibody variable region is determined by sequencing the antibody gene within the phage genome (Arap et al., Science 1998, 279:377-380; Smith et al., Science 1985, 228:1315-1317).

[0267] In specific embodiments, the present invention uses recombinant proteins of the HER2 extracellular domain as target antigens for immunizing mice. Gene fragments encoding the heavy chain variable region family and light chain variable region family of the antibody are isolated from spleen cells of immunized mice, and scFv fragments are then prepared and inserted into the phage surface protein III gene. Through co-expression, these fragments participate in phage assembly and are presented on the phage surface, thus constructing a phage display library. Using the phage display library, multiple adsorption-elution-amplification (panning) cycles are performed against a specific target antigen (e.g., the antigen used to immunize mice) to enrich phages that can specifically bind to the target antigen. Then, corresponding DNA sequence information is obtained via gene sequencing techniques, from which the amino acid sequence of the antibody variable region is further estimated.

[0268] 5. Preparation method 5.1 Method for preparing anti-HER2 antibodies or their antigen-binding fragments, or anti-HER2 biparatopic antibodies The present invention provides a method for preparing the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 biparatopic antibody, using the above-mentioned host cells.

[0269] This method comprises transfecting host cells with the anti-HER2 antibody of the present invention or its antigen-binding fragment, or a nucleic acid or expression vector encoding the anti-HER2 biparatopic antibody of the present invention, and culturing the host cells in a culture medium for a certain period of time to express the anti-HER2 antibody of the present invention or its antigen-binding fragment, or the anti-HER2 biparatopic antibody of the present invention. A commercially available culture medium may be used as the culture medium, but is not limited to this method.

[0270] Preferably, the expressed anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 biparatopic antibody, may be secreted into the culture medium in which the host cells are cultured. The antibody is recovered from the culture medium using conventional protein purification methods such as centrifugation or ultrafiltration to remove impurities, or affinity chromatography to purify the resulting product, although other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography may also be used.

[0271] 5.2 Preparation method for anti-HER2 biparatopic ADCs The ADC of the present invention can be prepared by any method known in the art, including but not limited to the following: (1) reacting the nucleophilic or electrophilic group of an antibody with a cleavable linker to form an antibody cleavable linker intermediate (Ab-L) via covalent bonding, and subsequently reacting it with a small molecule toxin compound (D) (wherein intermediate Ab-L may or may not undergo a purification step before reacting with the small molecule toxin compound); (2) reacting the nucleophilic or electrophilic group of a small molecule toxin compound with a cleavable linker to form a small molecule toxin compound cleavable linker intermediate (DL) via covalent bonding, and subsequently reacting it with the nucleophilic or electrophilic group of an antibody (wherein intermediate DL may or may not undergo a purification step before reacting with the antibody); or (3) simultaneously mixing and reacting the antibody, the cleavable linker, and the small molecule toxin compound to simultaneously form covalent bonds between the antibody and the cleavable linker and between the cleavable linker and the small molecule toxin compound, thereby preparing the ADC of the present invention. Several specific examples of methods for preparing ADCs, such as those described in U.S. Patent No. 8,624,003 (one-pot method), U.S. Patent No. 8,163,888 (one-step method), and U.S. Patent No. 5,208,020 (two-step method), are known in the art.

[0272] In some embodiments, the antibody is subjected to reducing conditions before the binding reaction to generate one or more free cysteine ​​residues. In some embodiments, a reducing agent (e.g., dithiothreitol [DTT], 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine [TCEP]) is used to interact with the antibody to reduce the interchain disulfide bonds in the hinge region of the antibody, thereby generating a partially or completely reduced antibody with free sulfhydryl groups. The reducing agent preferentially reduces the interchain disulfide bonds in the hinge region of the antibody while leaving the intrachain disulfide bonds of the antibody intact. In some embodiments, the antibody is reacted with the reducing agent TCEP in a buffer containing a chelating agent to obtain a partially or completely reduced antibody with free sulfhydryl groups. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), and examples of buffers include, but are not limited to, histidine hydrochloride, sodium phosphate, sodium borate, and sodium acetate solutions.

[0273] In some embodiments, a partially or completely reduced antibody having the free sulfhydryl group described above can react with a reactive functional group (e.g., a 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), thereby yielding an antibody cleavable linker intermediate (Ab-L) or ADC. Hereinafter, the terms “small molecule toxin compound-cleavable linker intermediate (DL)” are used interchangeably with “drug payload” or “payload.”

[0274] The purification method for ADCs prepared by the procedure described above may be any biochemical technique known in the art for protein purification or any combination thereof, for example, 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.

[0275] 6. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising the anti-HER2 antibody or its antigen-binding fragment, the anti-HER2 biparatopic antibody of the present invention, or the anti-HER2 biparatopic ADC of the present invention, and a pharmaceutically acceptable carrier.

[0276] Pharmaceutical compositions may contain any type of pharmaceutically acceptable carrier. Suitable carriers include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. For example, the selection and use of suitable carriers is described in Gennaro, Ed. Remington: The Science and Practice of Pharmacy, 20th Edition, 2003 (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.

[0277] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration. As used herein, the term “parenteral administration” refers to non-enteral and topical administration methods, including but not limited to injections and infusions into intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal regions. Alternatively, the antibodies of the present invention may also be administered by non-injection routes (e.g., topical, epidermal, or mucosal administration routes), such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. Considering different administration routes, the active ingredient may be encapsulated in a material to protect it from the effects of acids / bases and other natural conditions that may inactivate it.

[0278] Pharmaceutical compositions may be sterile aqueous solutions or dispersions. They can also form microemulsions, liposomes, or other combinations of ordered structures suitable for high drug concentrations.

[0279] The amount of active ingredient that can be combined with a carrier material to form a single dosage form is determined based on the target and the specific mode of administration, and is generally the amount of a composition that can produce a therapeutic effect. Typically, compositions formed using pharmaceutically acceptable carriers contain about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, and most preferably about 1% to about 30%.

[0280] The administration regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, several divided doses may be administered, or the dose may be proportionally reduced or increased depending on the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the composition for parenteral administration into dose units. As used herein, “dose unit” refers to a physically distinct unit suitable as a unit dose for treating a target, and each unit dose contains a predetermined amount of active ingredient obtained by calculating the amount of active ingredient to be administered co-administered with the pharmaceutical carrier required to produce the desired therapeutic effect. Furthermore, the anti-HER2 antibody or its antigen-binding fragment, anti-HER2 biparatopic antibody, or anti-HER2 biparatopic ADC of the present invention can also be administered as a sustained-release formulation, thereby reducing the frequency of administration.

[0281] The present invention provides for an anti-HER2 antibody or its antigen-binding fragment, an anti-HER2 biparatopic antibody or anti-HER2 biparatopic ADC, or a composition comprising an anti-HER2 antibody or its antigen-binding fragment, an anti-HER2 biparatopic antibody or anti-HER2 biparatopic ADC, which can be administered in doses ranging from approximately 0.0001 mg / kg to 100 mg / kg body weight, typically from 0.001 mg / kg to 50 mg / kg body weight.

[0282] The “therapeutic effective dose” of the anti-HER2 antibody or its antigen-binding fragment, anti-HER2 biparatopic antibody, or anti-HER2 biparatopic ADC of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of progression-free intervals, or prevention of physical impairment or disability caused by the disease. For example, the “therapeutic effective dose” for a subject with a tumor 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% compared to an untreated subject. A therapeutic effective dose of the therapeutic antibody or ADC can reduce the tumor size of a subject or alleviate symptoms, and the subject is typically a human or other mammal. The “therapeutic effective dose” can also be determined differently based on various factors including, but are not limited to, the method of formulation, method of administration, the patient’s age, physical condition, weight, sex or pathological condition, diet, administration time, administration interval, excretion rate and response sensitivity.

[0283] The pharmaceutical composition may be selected from controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, 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).

[0284] Therapeutic pharmaceutical compositions can be delivered by medical devices selected from the group consisting of: (1) needleless subcutaneous injection devices (e.g., U.S. Patent No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 and 4,596,556), (2) microinfusion pumps (U.S. Patent No. 4,487,603), (3) transdermal devices (U.S. Patent No. 4,486,194), (4) infusion sets (U.S. Patents No. 4,447,233 and 4,447,224), and (5) osmotic devices (U.S. Patents No. 4,439,196 and 4,475,196), the disclosures thereof incorporated herein by reference.

[0285] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment, anti-HER2 biparatopic antibody, or anti-HER2 biparatopic ADC of the present invention can be formulated to ensure in vivo in vivo distribution. For example, to ensure that the therapeutic antibody or ADC of the present invention crosses the blood-brain barrier, the therapeutic antibody or ADC can be formulated into liposomes, and the liposomes may further contain a targeted moiety to enhance selective delivery to specific cells or organs. For example, 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. 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. al., J Biol Chem 1994, 269:9090-9098, Keinanen and See Laukkanen, FEBS Lett 1994, 346:123–126 and Killion and Fidler, Immunomethods 1994, 4:273–279.

[0286] 7. Kit The present invention provides a kit comprising an effective amount of the anti-HER2 antibody or its antigen-binding fragment of the present invention, the anti-HER2 biparatopic antibody of the present invention, the anti-HER2 biparatopic ADC of the present invention, or the pharmaceutical composition of the present invention, and optionally at least one further oncological agent (i.e., the kit may or may not include at least one additional oncological agent). Preferably, the oncological agent may include, but is not limited to, another antagonist of ErbB2 / HER2; EGFR antagonists, HER3 antagonists; MET antagonists, small molecule inhibitors of MET (e.g., capmatinib); IGF1R antagonists (e.g., anti-IGF1R antibody); B-Raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720); PDGFR-α inhibitors (e.g., anti-PDG FR-α antibodies); PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors [e.g., imatinib mesylate or sunitinib maleate]); PDGF ligand inhibitors (e.g., anti-PDGF-A antibodies, anti-PDGF-B antibodies, anti-PDGF-C antibodies, anti-PDGF-D antibodies, aptamers, siRNA, etc.); VEGF antagonists (e.g., VEGF-Trap, e.g., affilibercept); 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., anti-Ang2 antibody disclosed in WO2011014469, H1H685P, etc.); FOLH1 antagonists (e.g., anti-FOLH1 antibody); STEAP1 or STEAP2 antagonists; TMPRSS2 antagonists; MSLN antagonists; MUC16 antagonists Agonists; CLEC12A antagonists; PD-1 or PD-L1 inhibitors (e.g., pembrolizumab or nivolumab); hormone receptor modulators (e.g., estrogen receptor modulators [tamoxifen, etc.]); androgen receptor modulators); aromatase inhibitors (e.g., letrozole, anastrozole, exemestane); kinase inhibitors (e.g., tyrosine kinase inhibitors [lapatinib, etc.]); 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, and IL-18, or their receptors); chemotherapeutic agents (including, but not limited to, microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, etc.).

[0287] In some embodiments, another antagonist of ErbB2 / HER2 includes anti-HER2 monoclonal antibodies (e.g., trastuzumab, pertuzumab) and / or ADCs (e.g., T-DM1, DS-8201), or anti-ErbB2 / HER2 small molecule inhibitors (e.g., tyrosine kinase inhibitors, e.g., lapatinib, pirotinib, and neratinib), and the EGFR antagonist includes anti-EGFR antibodies (e.g., cetus). This includes simab (panitumumab), anti-EGFR small molecule inhibitors (e.g., gefitinib, erlotinib), and anti-EGFRvIII antagonists (e.g., anti-EGFRvIII antibodies), and HER3 antagonists, but not limited to anti-HER3 antibodies (e.g., patritumab), and MET antagonists, but not limited to anti-MET antibodies (e.g., onartuzumab, emibetuzumab, and H4H14639D).

[0288] 8. Methods and uses for the treatment of HER2-expressing cancer In one embodiment, the present invention relates to a method for treating HER2-expressing cancer, comprising administering an effective amount of an anti-HER2 biparatopic antibody, an anti-HER2 biparatopic ADC, or a pharmaceutical composition or kit of the present invention to a subject in need thereof. Alternatively, the present invention relates to the use of the above-mentioned antibody, ADC, pharmaceutical composition or kit in the preparation of a pharmacopoeia for the treatment of HER2-expressing cancer. Alternatively, this application relates to the above-mentioned antibody, ADC, pharmaceutical composition or kit for use in the treatment of HER2-expressing cancer.

[0289] Cancer includes, but is not limited to, breast cancer, ovarian cancer, cervical cancer, colorectal cancer, stomach 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. Cancer also includes all stages of cancer, such as early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. Cancer may be HER2 overexpression or HER2 low expression. Subjects may be humans, non-human primates, or other mammals such as dogs, mice, or rats.

[0290] As is known in the art, HER2-expressing cancers can be characterized by the level of HER2 expression on the surface of cancer cells (i.e., the "HER2 state"). HER2 expression levels can be assessed using methods such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In some embodiments, the cancer is a HER2-overexpressing cancer (e.g., IHC3+ or IHC2+ / FISH). + ) and / or HER2-low expression cancer (IHC2+ / FISH) - Includes (or IHC1+).

[0291] In some embodiments, anti-HER2 biparatopic ADCs or their pharmaceutical compositions exhibit cytotoxic effects against both HER2-overexpressing tumors / cancers and HER2-low-expressing tumors / cancers.

[0292] In some embodiments, the anti-HER2 biparatopic antibody or its pharmaceutical composition has inhibitory activity against the growth or proliferation of HER2-overexpressing tumor cells.

[0293] In some embodiments, administration of an effective amount of the anti-HER2 biparatopic antibody, anti-HER2 biparatopic ADC, pharmaceutical composition, or kit of the present invention to a subject in need can reduce the tumor volume of the subject, inhibit tumor growth, extend the subject's disease-free or progression-free survival rate, increase the subject's overall survival rate, reduce tumor metastasis, or improve the subject's quality of life.

[0294] In another aspect, the present invention also relates to a method for treating cancer that has become resistant to or has relapsed to existing HER2-targeted therapies, comprising administering an effective amount of the present invention's anti-HER2 biparatopic antibody, anti-HER2 biparatopic ADC, pharmaceutical composition, or kit to a patient in need thereof. Alternatively, the present invention relates to the use of an antibody, ADC, pharmaceutical composition, or kit in the preparation of a pharmacopoeia for treating cancer that has become resistant to or has relapsed to HER2-targeted therapies.

[0295] In some embodiments, patients exhibit a non-response or inadequate response to one or more existing HER2-targeted therapies, including trastuzumab, pertuzumab, T-DM1, and DS-8201. Non-response may manifest as tumor growth, increased tumor volume, metastasis formation, or an increase in the number of metastases in the patient. Non-response may also manifest as a shortened time to metastasis development or disease progression. Poor response refers to tumor growth or metastasis occurring in the patient during or immediately after standard treatment with a HER2-targeted therapy.

[0296] In another aspect, the present invention also relates to a method for treating patients who are ineligible for or refractory to existing HER2-targeted therapies, or who have acquired resistance to or relapsed to existing HER2-targeted therapies, comprising administering an effective amount of the present invention's anti-HER2 biparatopic antibody, anti-HER2 biparatopic ADC, pharmaceutical composition, or kit to patients in need thereof. Alternatively, the present invention relates to the use of an antibody, ADC, pharmaceutical composition, or kit in the preparation of a medicament for treating patients who are ineligible for or refractory to existing HER2-targeted therapies, or who have acquired resistance to or relapsed to existing HER2-targeted therapies. HER2-targeted therapies include treatment with trastuzumab, pertuzumab, T-DM1, or DS-8201.

[0297] In some embodiments, the anti-HER2 biparatopic antibody, anti-HER2 biparatopic ADC, pharmaceutical composition, or kit of the present invention can be used to treat advanced cancer in patients who have experienced disease progression after receiving existing HER2-targeted therapy.

[0298] In another embodiment, the anti-HER2 biparatopic antibody, anti-HER2 biparatopic ADC, pharmaceutical composition, or kit 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, and radiofrequency ablation.

[0299] 9. Use for detection The present invention also relates to the use of an anti-HER2 antibody or its antigen-binding fragment or an anti-HER2 biparatopic antibody for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, and for screening cancer patients responsive to treatment with the anti-HER2 biparatopic ADC of the present invention. Alternatively, this 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 responsive to the aforementioned ADC treatment, comprising incubating an anti-HER2 antibody or its antigen-binding fragment or an anti-HER2 biparatopic antibody with a sample or biological specimen isolated from a patient and detecting whether the antibody binds to the sample or biological specimen. Alternatively, this application relates to the aforementioned anti-HER2 antibody or its antigen-binding fragment or an anti-HER2 biparatopic antibody used for detecting and / or measuring HER2 or HER2-expressing tumor cells in a sample, or for screening cancer patients responsive to the aforementioned ADC treatment.

[0300] In some embodiments, anti-HER2 antibodies or their antigen-binding fragments, or anti-HER2 biparatopic antibodies, can be used to diagnose conditions or diseases associated with abnormal HER2 expression (e.g., overexpression, underexpression, or absence of expression) to facilitate the determination of treatment regimens. For example, the antibody can be conjugated with a detectable label or reporter molecule, and the labeled antibody can be incubated with a sample obtained from the patient to diagnose and determine HER2 expression. The detectable label or reporter molecule is: 3 H, 14 C, 32 P, 35 S or 125 These may be radioactive isotopes such as I, fluorescein, rhodamine, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, chemiluminescent materials such as luminol, bioluminescent substances such as luciferase, luciferin, aequorin, or enzymes such as alkaline phosphatase, β-galactosidase, acetylcholinesterase, horseradish peroxidase, luciferase. Specific exemplary assays that can be used to detect or measure HER2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immuno-PET (e.g., 89 Zr, 64 Examples include the use of Cu, etc., and fluorescence-activated cell sorting (FACS).

[0301] The present invention will be further illustrated by the following examples, which should not be construed as further limitations. All drawings and all references, patents and patent applications cited throughout the present invention are expressly incorporated herein by reference. Unless otherwise specified, the materials, reagents and equipment included in the following examples are commercially available. [Examples]

[0302] Example 1: Construction of an anti-HER2 antibody library and screening of antibodies 1.1 Construction of an anti-HER2 antibody library Two BALB / c mice were immunized with recombinant human HER2 extracellular domain protein (sequence derived from positions 23-652 of NP_004439.2). After three immunizations, blood was collected from the mice and antibody titers were measured by ELISA. The results showed that titers reached levels exceeding 1:1,000,000 (data not shown). Subsequently, the mice were given booster immunization, and after 3 days, their spleens were aseptically isolated, and single splenocyte suspensions were prepared from each spleen. The recovered mouse splenocytes were lysed, and total RNA was extracted using the Animal Total RNA Isolation Kit (FuGene). The extracted RNA was used as a reverse transcription template using a reverse transcription kit (PrimeScript® II First Strand cDNA Synthesis Kit, Takara) to produce cDNA. The cDNA was then used as a template for PCR with specific primers to amplify the complete repertoire of mouse antibody gene fragments encoding the heavy chain variable region and light chain variable region. Light chain and heavy chain variable region gene fragments were spliced ​​and amplified by subdivision extension PCR (SOE-PCR) to generate single-chain antibody variable region (scFv) gene fragments. The scFv sequences were digested, ligated to a phagemide display vector, and the ligated product was electroporated into TG1 competent cells. After infecting the cell cultures with helper phage M13KO7, two phage display libraries were obtained.

[0303] 1.2 Screening and Sequence Analysis of Anti-HER2 Antibodies Using recombinant HER2 extracellular domain protein as the target antigen, two phage display libraries described in Section 1.1 of this example were panned. After 3 to 5 "adsorption-elution-amplification" panning cycles, followed by colony ELISA screening and PCR digestion validation, and subsequent sequencing analysis of candidate clones, 15 sequence-specific anti-HER2 mouse monoclonal antibodies were ultimately obtained.

[0304] 1.3 Preparation of anti-HER2 chimeric antibodies The light and heavy chain genes of the 15 mouse anti-HER2 antibodies listed above were homologously recombined with the linearized expression vector pcDNA3.1 (the heavy chain variable region VH was ligated to a pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region VL was ligated to a pcDNA3.1 vector containing the human CK constant region). Sequence-confirmed chimeric antibody expression vectors were obtained by colony PCR and DNA sequencing. The chimeric antibody expression vectors were extracted and purified using conventional methods. Next, the 15 groups of chimeric antibody expression vectors containing the light and heavy chain gene segments were transiently transfected into ExpiCHO-S cells (Thermo). The anti-HER2 chimeric antibodies were expressed in serum-free cultures and then purified by protein A affinity chromatography using the AKTA Pure system.

[0305] 1.4 Detection of the binding activity of anti-HER2 chimeric antibodies against ErbB / HER family members The binding activity of anti-HER2 chimeric antibodies to human ErbB / HER family members was detected by ELISA. The specific procedure was as follows: Recombinant proteins of the extracellular domain of human HER2 and other human ErbB / HER family members (EGFR, HER3, and HER4) were diluted in PBS and then added to a 96-well ELISA plate at a rate of 100 μL / well, followed by incubation at 4°C overnight. After washing the plate with PBS, 200 μL of blocking solution (PBST containing 1% BSA [PBS + 0.1% Tween-20]) was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of test antibodies (anti-HER2 chimeric antibody and control antibodies trastuzumab, cetuximab, or patrizumab), serially diluted with blocking buffer, was added to each well and the plate was incubated at room temperature for 2 hours. After washing the plates with PBST, 100 μL of anti-human IgG(H+L)-HRP antibody (Jackson Immuno), diluted 1:10,000 with blocking buffer, was added to each well and incubated at room temperature for 1 hour. After washing the plates with PBST again, 100 μL of TMB substrate solution was added to each well and incubated at room temperature in the dark for 3–10 minutes. Then, 50 μL of stop solution (2M HCl) was added to each well, and the OD450 values ​​were read using a multimode microplate reader (Varioskan, Thermo). The raw data were analyzed using GraphPad Prism9 software. As shown in Figure 1, mAb2117, mAb2126, mAb2128, mAb2133, mAb2138, mAb2164, and mAb2170 showed strong binding activity to HER2 and no cross-reactivity with other human ErbB / HER family members.

[0306] 1.5 Detection of the binding activity of anti-HER2 chimeric antibodies against HER2-expressing tumor cells The binding activity of anti-HER2 chimeric antibodies to NCI-N87 cells was detected by flow cytometry. The detailed procedure was as follows: NCI-N87 cells were harvested, washed once with ice-cold FACS buffer (PBS + 1% BSA), and then 6-10 × 10⁻¹⁰⁻¹ 6 The cells were resuspended in cells / mL, and 50 μL of the cell suspension was added to each well of a 96-well U-bottom plate. Subsequently, 50 μL of serially diluted test chimeric antibody or positive control antibody trastuzumab (including FACS buffer as a negative blank control) was added per well, and the plate was incubated on ice for 60 minutes. After washing the cells twice with pre-cooled FACS buffer, 100 μL / well of anti-human IgG(H+L)-AF488 antibody (Jackson Immuno), diluted 1:1,000 with FACS buffer, was added, and the plate was incubated on ice in the dark for 40 minutes. After washing the cells twice with pre-cooled FACS buffer, the cells were resuspended in 100 μL of pre-cooled FACS buffer and analyzed using a flow cytometer (NovoCyte3005, Agilent). As shown in Figure 2, antibodies mAb2117, mAb2126, mAb2164, and mAb2170 showed strong binding activity to NCI-N87 cells.

[0307] 1.6 Analysis of antigen-binding epitopes of anti-HER2 chimeric antibodies Based on the structures of human and mouse HER2 extracellular domain subdomains, gene sequences encoding different subdomains of human and mouse HER2 were recombinantly spliced ​​and ligated to a pcDNA3.1 vector via homologous recombination to construct expression plasmids for human-mouse chimeric HER2 extracellular recombinant proteins (Figure 3A). Transient transfection was performed to prepare supernatants containing human-mouse chimeric HER2 extracellular domain recombinant proteins (each recombinant protein contained a 6×His tag at its C-terminus). Using the human-mouse chimeric recombinant proteins as antigens, the binding epitopes of chimeric antibodies within the HER2 extracellular domain were detected by ELISA. The specific method was as follows: the test chimeric antibody was diluted with PBS and added to a 96-well ELISA plate at 100 μL / well, followed by incubation at 4°C overnight. After washing the plate with PBS, 200 μL of blocking buffer (PBST containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour. After washing the plates with PBST, 100 μL of human HER2, mouse HER2, or human-mouse chimeric HER2 recombinant protein, diluted 1:200 with blocking buffer, was added to each well. The chimeric proteins included HER2(D1), HER2(D1-2), HER2(D1-3), HER2(D2), HER2(D3), and HER2(D4), where the subscript in parentheses indicates that one or more extracellular subdomains of the HER2 chimeric antigen were replaced by the corresponding mouse sequence. For example, D1 indicates that extracellular subdomain 1 was replaced with 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 with blocking buffer, was added to each well and incubated at room temperature for 1 hour. After washing the plates with PBST, 100 μL of TMB substrate solution was added to each well, and the plates were incubated at room temperature in the dark for 3–10 minutes. Then, 50 μL of stop solution (2M HCl) was added to each well, and the OD450 values ​​were read using a multimode microplate reader (Varioskan, Thermo).The raw data was analyzed using GraphPad Prism9 software. As shown in Figure 3B, the epitope recognized by mAb2117 is located at D3 of the HER2 extracellular domain, the epitopes recognized by mAb2126, mAb2164, and mAb2170 are located at D1 of the HER2 extracellular domain, and the epitope recognized by mAb2128 is located at D4 of the HER2 extracellular domain.

[0308] 1.7 Analysis of antigen binding competition among anti-HER2 chimeric antibodies To further investigate whether epitopes bound by chimeric antibodies on HER2 overlap or cross-react, competitive antigen-binding activity between chimeric antibodies was detected by competitive ELISA. The specific procedure was as follows: Recombinant HER2 extracellular domain protein was diluted with PBS and added to a 96-well ELISA plate at a rate of 100 μL / well, followed by incubation at 4°C overnight. After washing the plate 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 of the test chimeric antibody or control antibody trastuzumab, serially diluted with blocking buffer (starting at 50 μg / mL, followed by 3-fold serial dilutions for a total of 7 concentration gradients), was added to each well. After incubation at room temperature for 1 hour, 50 μL of biotin-labeled chimeric antibody (all at a final concentration of 0.5 μg / mL) was added to each well and incubated at room temperature for a further 1 hour. After washing with PBST, 100 μL of HRP-streptavidin (Jackson Immuno), diluted 1:10,000 with blocking buffer, was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of TMB substrate 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 the OD450 value was measured using a multimode microplate reader (Varioskan, Thermo). The results showed that mAb2164 and mAb2170 completely competed for binding of the HER2 extracellular domain recombinant protein (Figure 4A), mAb2164 partially competed with mAb2117 and mAb2126 for binding of the HER2 extracellular domain recombinant protein, but mAb2164 did not compete with trastuzumab for binding of the HER2 extracellular domain recombinant protein (Figure 4A), mAb2164 did not compete with mAb2128 for binding of the HER2 extracellular domain recombinant protein, and mAb2117 did not compete with mAb2126 or mAb2128 for binding of the HER2 extracellular domain recombinant protein (Figure 4B).

[0309] 1.8 Analysis of antigen-binding dynamics of anti-HER2 chimeric antibodies The binding dynamics of each chimeric antibody to recombinant HER2 extracellular domain protein were determined using a molecular interaction analyzer (ForteBio, Model R8). The specific procedure was as follows: Test chimeric antibodies (mAb2117, mAb2126, mAb2128, mAb2164, and mAb2170) were diluted to 5 μg / mL in PBS and added to 96-well black-wall plates at 200 μL / well. Recombinant HER2 extracellular domain protein was diluted to 200 nM in PBS, subjected to 1:2 serial dilution, and then added to 96-well black-wall plates at 200 μL / well. The 96-well black-wall plates containing the test samples and protein A sensors were placed in the analyzer. The program was set so that the protein A sensor would capture the antibody and subsequently detect the binding and dissociation interactions with the antigen. After each binding and dissociation cycle, the sensor was regenerated by immersion in 10 mM glycine buffer (pH 1.5) before the next cycle. After detection, the binding rate constant and dissociation rate constant of the antibody and antigen are fitted and extrapolated using analysis software, and the affinity constant (K D The value of ) was calculated. As shown in Table 4, the binding affinity constant (K) of chimeric antibodies (mAb2117, mAb2126, mAb2128, mAb2164 and mAb2170) against recombinant HER2 extracellular domain protein was calculated. D ) are all approximately 10 -9 It was M.

[0310] [Table 4]

[0311] 1.9 Effect of anti-HER2 chimeric antibody on HER2 phosphorylation at Y1248 Published studies have demonstrated that trastuzumab can induce phosphorylation of the intracellular domain of HER2 at Y1248 in cardiomyocytes, ultimately triggering a series of signaling cascades that disrupt cardiomyocyte homeostasis (Mohan et al., Molecular Cancer Therapeutics 2016, 15:1321-1331), which may be one of the underlying mechanisms of trastuzumab-induced cardiotoxicity. Therefore, in this example, we evaluated whether anti-HER2 antibodies possess trastuzumab-like cardiotoxicity by detecting their ability to induce phosphorylation of HER2-Y1248 in SKBR-3 cells. The specific method is as follows: SKBR-3 cells were harvested, resuspended in serum-free RPMI-1640 medium (containing 0.1% BSA), seeded in 6-well plates, and then cultured overnight at 37°C. Test antibodies or control antibodies (trastuzumab and pertuzumab) were 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 in 1.5 mL EP tubes, washed twice with pre-cooled PBS, and then dissolved in 150 μL of RIPA cell lysis buffer (Porteintech) to extract total protein. The cell lysis supernatant was collected by high-speed centrifugation at 4°C for 10 minutes. The protein concentration in the supernatant was determined by BCA assay, and samples for protein electrophoresis were prepared by adding reducing buffer, followed by SDS-PAGE gel electrophoresis. After electrophoresis, the protein bands were transferred to a PVDF membrane at 80 V for 90 minutes. The PVDF membrane was 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 dilution of 1:1000 and incubated overnight at 4°C. After washing the PVDF membrane three times with PBST, goat anti-rabbit IgG-HRP antibody (Jacskon Immuno), diluted 1:5000 with 5% skim milk, was added and incubated at room temperature for 1 hour. After washing the PVDF membrane three times with PBST again, developer was added and detection was performed using a Bio-Rad imaging system.As shown in Figure 5, compared to the positive control trastuzumab, the chimeric antibodies mAb2117 and mAb2126 do not induce phosphorylation of HER2-Y1248 and may not have trastuzumab-like cardiotoxicity.

[0312] 1.10 Effect of anti-HER2 chimeric antibodies on NRG-1-induced activation of the HER2 downstream signaling pathway NRG-1 can induce HER2 heterodimerization and activate downstream signaling pathways, with AKT phosphorylation being a prominent event in this signaling cascade. In this example, the effect of an antibody on HER2 receptor signaling was evaluated by assessing its inhibitory activity against NRG-1-induced AKT phosphorylation. The specific procedure was as follows: T47D cells were seeded in a 6-well plate and cultured until approximately 80% confluence was reached. The medium was removed and replaced with serum-free RPMI-1640 for overnight starvation. The test antibody or control antibody (trastuzumab, pertuzumab) was added to each well at a final concentration of 100 nM and incubated at 37°C for 20 minutes. Then, NRG-1 (NOVUS) was added to each well at a final concentration of 100 ng / mL and incubated for a further 10 minutes. After washing the cells twice with pre-cooled PBS, total protein extraction and Western blot analysis were performed as described in Section 1.9 of this example. The primary antibody used was either 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 (Jackson Immuno). As shown in Figure 6, compared to the positive control antibody pertuzumab, the chimeric antibodies mAb2117 and mAb2126 did not have an inhibitory effect on NRG-1-induced AKT phosphorylation, indicating that they did not interfere with NRG-1-induced HER2 heterodimerization and the activation of its downstream signaling pathway.

[0313] Example 2: Humanization and optimization of the anti-HER2 chimeric antibody mAb2117 2.1 Humanization of the anti-HER2 chimeric antibody mAb2117 Based on analysis using MOE software, the heavy chain framework region sequence of the chimeric antibody mAb2117 was replaced with the framework region sequence of Germline IGHV3-23 by CDR grafting and named Hu2117-H1, and the light chain framework region sequence of mAb2117 was replaced with the framework region sequence of Germline IGKV1-39 and named Hu2117-K1. The chimeric antibody mAb2117 and its humanized antibody were constructed as single-chain antibody fusion proteins with hIgG1 Fc (scFv-hIgG1 Fc fusion proteins). The scFv antibody gene fragments, containing humanized single-chain antibodies Hu2117HK and Hu2117KH, and Mu2117HK and Mu2117KH chimeric single-chain control antibodies, were synthesized by gene synthesis. Here, "HK" represents a single-chain antibody structure arranged as "(N-terminal) heavy chain variable region nucleic acid sequence - linker (G4S)3 - light chain variable region nucleic acid sequence (C-terminal)," and "KH" represents a single-chain antibody structure arranged as "(N-terminal) light chain variable region nucleic acid sequence - linker (G4S)3 - heavy chain variable region nucleic acid sequence (C-terminal)." The scFv gene fragments were homologously rearranged in a pcDNA3.1 vector (containing human IgG1 Fc sequence) to construct antibody expression plasmids. After extracting the expression plasmids, they were transiently transfected into ExpiCHO-S cells (Thermo) for serum-free culture. The antibody was purified from the culture supernatant by protein A affinity chromatography using the AKTA Pure system to obtain a single-chain antibody fusion protein. The binding dynamics of the single-chain antibody fusion protein to recombinant HER2 protein were analyzed using ForteBio. As shown in Table 5, the results showed that the humanized antibody retained the binding activity of its parent antibody to HER2 and maintained an affinity constant (K D ) is about 10 -7 It was proven that it is M.

[0314] [Table 5]

[0315] The binding activity of single-chain antibody fusion proteins Hu2117HK and Hu2117KH to BT474 cells was detected using flow cytometry according to the method described in Section 1.5 of Example 1, with the chimeric antibody mAb2117 and trastuzumab used as positive controls. The results demonstrated that mAb2117, Hu2117HK, and Hu2117KH could all bind to BT474 cells, with the binding activity of Hu2117HK being relatively higher than that of Hu2117KH (data not shown).

[0316] 2.2 Optimization of the humanized anti-HER2 antibody Hu2117HK by single or multipoint mutations Using the online software abYsis (http: / / abysis.org / abysis / index.html) and the software MOE, we identified low-humanity levels of amino acid residues, potential stability-responsible sites, and mouse-derived amino acid residues in the framework region within the light chain variable region sequence and heavy chain variable region sequence of the humanized antibody Hu2117HK. To improve the humanization, antigen-binding affinity, and / or stability of the antibody, a total of 50 single-point or multi-point mutations were designed. Specifically, each of the 50 designed single-point or multi-point mutations was introduced into the sequence of the single-chain antibody Hu2117HK by PCR, and the mutant protein of the single-chain antibody was expressed.

[0317] The transient expression levels of these 50 mutant single-chain antibodies and their parent single-chain antibody Hu2117HK were detected using a ForteBio Molecular Interaction Analyzer. The specific procedure was as follows: After transient expression in ExpiCHO-S cells, the culture supernatant was collected and added to a 96-well black-wall plate at 200 μL / well. The test samples and protein A sensors were placed in the ForteBio analyzer, and the binding rate of each antibody molecule in the culture supernatant to the protein A sensor was measured using a pre-configured quantitative program. After each cycle, the sensors were regenerated by immersion in 10 mM glycine buffer (pH 1.5) before the next cycle. After detection, the expression level of each single-chain antibody was calculated based on a standard curve using analysis software. The binding dynamics of each single-chain antibody variant to HER2 were also analyzed using ForteBio according to the method described in Section 1.8 of Example 1. The results for single / multipoint mutation information, mutant amino acids, transient expression levels, and antigen binding dynamics are shown in Table 6.

[0318] [Table 6-1] [Table 6-2]

[0319] The binding activity of single-chain antibodies containing one-point / multipoint mutations against BT474 cells and RT-112 cells was detected by flow cytometry according to the method described in Section 1.5 of Example 1, with the parental single-chain antibody Hu2117HK used as a positive control. As shown in Figure 7, compared to the parental single-chain antibody Hu2117HK, most single-chain antibody variants retained their binding activity against BT474 cells, including Hu2117HK-Mu01, -Mu02, -Mu03, -Mu04, -Mu05, -Mu06, -Mu08, -Mu09, -Mu10, -Mu11, -Mu12, -Mu13, -Mu17, -M Several antibodies, including -Mu20, -Mu21, -Mu23, -Mu24, -Mu25, -Mu27, -Mu28, -Mu32, -Mu33, -Mu36, -Mu37, -Mu38, -Mu39, -Mu40, -Mu45, -Mu46, -Mu47, -Mu48, -Mu49, and -Mu50, showed slightly improved cell binding activity (Figure 7A). Similarly, the results of binding to RT-112 cells are shown in Figure 7B. Compared to the parent single-chain antibody Hu2117HK, most single-chain antibody variants, including 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, retained their binding activity to RT-112 cells.

[0320] 2.3 Optimization of the anti-HER2 humanized antibody Hu2117HK by combinational mutations Based on the preferred single / multipoint mutations identified above, combination mutations were introduced into the variable region sequence of the humanized single-chain antibody Hu2117HK to construct antibody variants containing different combination mutations, including Hu2117-HK201 to -HK203 (abbreviated as HK201~HK203) and Hu2117-HK301 to -HK312 (abbreviated as HK301~HK312). Single-chain antibody samples were prepared according to the method described in Section 2.2 of this example, and the transient expression levels of each variant and the parent single-chain antibody Hu2117HK were detected using ForteBio. The results for the combination mutations and transient expression levels contained in each antibody variant are summarized in Table 7.

[0321] [Table 7]

[0322] The binding activity of single-chain antibodies containing combinational mutations and the parental single-chain antibody Hu2117HK to BT474 and RT-112 cells was detected using flow cytometry, according to the method described in Section 1.5 of Example 1. The results showed that at a saturated concentration of 10 μg / mL, antibodies HK201, HK202, and HK203 could bind to both BT474 and RT-112 cells with binding activity comparable to that of the parental antibody Hu2117HK. HK301, HK303, HK304, HK305, HK306, HK309, and HK310 were also able to bind to RT-112 cells with binding activity comparable to that of the parental antibody Hu2117HK (data not shown).

[0323] To further characterize the cell-binding activity of the combinedly optimized antibodies, HK203, HK303, HK304, HK309, and HK310 were selected and analyzed by flow cytometry according to the method described in Section 1.5 of Example 1. The test antibodies were serially diluted and then incubated with BT474 and RT-112 cells using the parental single-chain antibody Hu2117HK as a control. As shown in Table 8 and Figure 8, all optimized humanized antibodies were able to bind to BT474 and RT-112 cells, and their binding activity was comparable to that of the parental antibody Hu2117HK.

[0324] [Table 8]

[0325] Example 3 Humanization and optimization of the anti-HER2 chimeric antibody mAb2126 3.1 Humanization of the anti-HER2 chimeric antibody mAb2126 Based on analysis using MOE software, the heavy chain framework region sequence of the chimeric antibody mAb2126 was replaced with the framework region sequence of Germline IGHV4-4 or IGHV7-4 by CDR grafting, and named Hu2126-H1 and Hu2126-H2, respectively. The light chain framework region sequence of mAb2126 was replaced with the framework region sequence of Germline IGKV1-39, and named Hu2126-K1. Variable region gene fragments of the humanized antibody were synthesized, and the light chain and heavy chain fragments were separately ligated into the linearized expression vector pcDNA3.1 to construct antibody expression plasmids (with the heavy chain variable region VH inserted into pcDNA3.1 containing the human IgG1 constant region, and the light chain variable region VL inserted into pcDNA3.1 containing the human Cκ constant region). The expression plasmids were extracted using conventional methods and transiently transfected into ExpiCHO-S cells (Thermo) for serum-free culture. Humanized antibodies Hu2126-H1K1 and Hu2126-H2K1 in the culture supernatant were purified by protein A affinity chromatography using the AKTA Pure system.

[0326] The binding kinetics of each humanized antibody to HER2 were determined using ForteBio according to the method described in Section 1.8 of Example 1. As shown in Table 9, the CDR-grafted humanized antibodies retained their binding activity to HER2, with the binding affinity of Hu2126-H2K1 being equivalent to that of the parental chimeric antibody mAb2126.

[0327] [Table 9]

[0328] 3.2 Optimization of the anti-HER2 humanized antibody Hu2126-H2K1 by single-point mutation Using the online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, the light chain and heavy chain variable region sequences of the humanized antibody Hu2126-H2K1 were analyzed to identify amino acid residues with relatively low levels of humanization, potential stability-responsible sites, and mouse-derived amino acid residues within the framework region. A total of 63 single-point mutations were designed to improve the humanization, antigen-binding affinity, and / or stability of the antibody. Specifically, each of the 63 designed single-point mutations was introduced into the humanized antibody Hu2126-H2K1 sequence by PCR, and then expression plasmids were constructed. The light chain and heavy chain expression plasmids were transiently and co-transfected into ExpiCHO-S cells for antibody expression, yielding cell culture supernatants of 63 antibody variants containing the single-point mutations. Following the method described in Section 2.2 of Example 2, the expression levels and binding kinetics to HER2 of each antibody variant after 3 days of transient culture were detected using ForteBio. The results are summarized in Table 10. Compared to the parent antibody Hu2126-H2K1, 42 of the 63 designed point mutations were identified as 17 point mutations in the antibody light chain: 2126-Hu-L02, -L05, -L06, -L07, -L08, -L09, -L10, -L11, -L12, -L13, -L14, -L15, -L16, -L17 and -L18, -L19 and -L21, and 25 point mutations in the antibody heavy chain. The mutations included 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, and -H59, which did not significantly affect the antigen-binding affinity of the antibody.

[0329] [Table 10-1] [Table 10-2]

[0330] 3.3 Optimization of the anti-HER2 humanized antibody Hu2126-H2K1 by combination mutations Selected single-point mutations of the light chain described above were introduced into Hu2126-K1, and selected single-point mutations of the heavy chain described above were introduced into Hu2126-H2. Accordingly, light chain combined mutation expression plasmids and heavy chain combined mutation expression plasmids were constructed. The light chain and heavy chain expression plasmids were then paired and transiently transfected into ExpiCHO-S cells. After culture, expression, and purification, a total of 10 antibody variants named Hu2126-H2K1-L71-H72b-Mu10~-Mu19 (abbreviated as 7172b-Mu10~-Mu19) were obtained. The binding kinetics of the humanized antibodies containing the combined mutations to HER2 were determined using ForteBio according to the method described in Section 1.8 of Example 1.

[0331] Table 11 summarizes the combination mutations and antigen-binding dynamics of each antibody variant. The binding affinities of antibodies 7172b-Mu10, 7172b-Mu14, 7172b-Mu17, and 7172b-Mu19 to HER2 were significantly higher than those of the parent antibody Hu2126-H2K1.

[0332] [Table 11]

[0333] Example 4: Construction, optimization, and functional activity analysis of an anti-HER2 biparatopic antibody. 4.1 Construction of anti-HER2 biparatopic antibodies In this example, a pair of monospecific antibodies (Hu2117-HK304 and Hu2126-H2K1-L71-H72b-Mu14) that recognize distinct epitopes on the HER2 extracellular domain without competing for binding were selected for the construction of an exemplary biparatopic antibody. As shown in Figure 9, the biparatopic antibody has a DVD-IgG configuration, with the Fv domain consisting of the variable region sequence of Hu2117-HK304 and the IgG domain consisting of the sequence of Hu2126-H2K1-L71-H72b-Mu14. The heavy chain variable region of the Fv domain is fused to the heavy chain of the IgG domain via a (G4S)1 linker, and the light chain variable region of the Fv domain is fused to the light chain of the IgG domain via a (G4S)3 linker. To prevent the effector function mediated by the antibody's Fc region from potentially causing the death of non-target cells, the L234F / L235E / P331S mutation (EU numbered "TM mutation") was introduced into the Fc region of the biparatopic antibody to reduce binding to FcγR and C1q, thereby eliminating the Fc effector function. The constructed biparatopic antibody was named 04BS-109-WT. The 04BS-109-WT expression plasmid was transiently transfected into ExpiCHO-S cells for protein expression, and the antibody was purified by protein A affinity chromatography. During purification, the 04BS-109-WT bispecific antibody was observed to form a colloidal precipitate upon elution (using an eluate without NaCl), resulting in a turbid elution product. However, after the addition of 0.1M NaCl, the colloidal precipitate gradually dissolved, eventually yielding a clear antibody solution.

[0334] 4.2 Optimization of anti-HER2 biparatopic antibodies Based on analysis using MOE software, a series of mutations were designed in the HCDR2 sequence of the Fv domain of the biparatopic antibody 04BS-109-WT to improve the molecular stability of the bispecific antibody. The designed mutations are listed in Table 12 and named 04BS-1123-ST01 to -ST06. The mutations were introduced into the 04BS-109-WT sequence via PCR. The constructed expression plasmids of each bispecific antibody variant were transiently transfected into ExpiCHO-S cells and cultured in serum-free medium for antibody protein expression. The antibody expression levels in the culture supernatant were measured using ForteBio according to the method described in section 2.2 of Example 2. The results showed that the expression level of antibody 04BS-1123-ST03 was too low (<30 mg / L), while the expression levels of the other bispecific antibody variants exceeded 300 mg / L after 10 days of culture. After purification by protein A affinity chromatography, the antibodies were analyzed by SEC-HPLC, and the data showed that the monomer content of each bispecific antibody variant was close to 100%. However, significant differences in molecular stability were observed among the bispecific antibody variants during the purification process (shown in Table 12). After the elution step in protein A affinity chromatography, the sample solution of bispecific antibody 04BS-1123-ST06 remained clear regardless of whether the eluate contained 0.1M NaCl or not, suggesting that the molecular stability of 04BS-1123-ST06 was superior to that of the other variants and the parent antibody 04BS-109-WT.

[0335] [Table 12]

[0336] 4.3 Cell-based competitive binding assay of anti-HER2 humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 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 non-competitive binding properties to HER2 of their parent antibodies (chimeric antibodies mAb2117 and mAb2126), competitive binding assays were performed according to the method described in Section 1.5 of Example 1. Two optimized antibodies, serially diluted from 800 μg / mL, were each mixed with an equal volume of 8 μg / mL biotin-labeled Hu2117-HK304-06 antibody and then incubated with BT474 cells. Using AF488-labeled streptavidin (Jackson Immuno) as a secondary antibody, the competitive inhibitory activity of these two antibodies against HER2-expressing tumor cells was evaluated by flow cytometry. As shown in Figure 10, Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 do not compete for the binding of HER2 expressed on the cell surface.

[0337] 4.4 Analysis of the internalization and lysosomal transport / degradation efficiency of anti-HER2 biparatopic antibodies In this example, the internalization rates of the aforementioned exemplary anti-HER2 biparatopic antibodies in cells expressing different levels of HER2 (including BT474, JIMT-1, and RT-112 cells) were determined using flow cytometry. The specific method was as follows: Cells were harvested by trypsin treatment, washed with ice-cold FACS buffer, and then 6–10 × 10⁻⁶ cells. 6The cells were resuspended in 1 / mL. The cell suspension was transferred to 96-well U-bottom deep-well plates at 300 μL / well. Then, 300 μL of test antibody (final antibody concentration: 20 μg / mL) was added to each well, mixed, and incubated on ice for 30–60 minutes. After washing twice with ice-cold FACS buffer, the cells in each well were completely resuspended in 600 μL / well of ice-cold FACS buffer, and then the cell suspension was uniformly dispensed at 100 μL / well into five replica 96-well U-bottom plates. In the five replica plates, one plate was used as the control group and incubated on ice, while the other four plates were used as the experimental groups and incubated at 37°C. Each plate was then transferred to ice for cooling, and the internalization reaction was completed at 30 minutes, 1 hour, 2 hours, and 4 hours, respectively.

[0338] At the end of incubation, cells in each well were washed twice with ice-cold FACS buffer and fixed overnight at 4°C with 100 μL / well of fixative (2% paraformaldehyde). After washing twice with ice-cold FACS buffer, 100 μL / well of AF488-labeled anti-human IgG(H+L) antibody (Jackson Immuno), diluted 1:1000 with ice-cold FACS buffer, was added to each plate. Cells were resuspended and incubated on ice in the dark for 40 minutes. After washing twice with ice-cold FACS buffer, cells were resuspended in 100 μL / well of FACS buffer and analyzed by flow cytometry. Antibody internalization rate (expressed as a percentage) was calculated using the following formula: (MFI 対照 -MFI 実験 ) / MFI 対照×100%. As shown in Figure 11, compared to the control antibody trastuzumab and the corresponding monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14, the anti-HER2 biparatopic antibodies 04BS-1123-ST04, 04BS-1123-ST05, 04BS-1123-ST06 and their parent antibody 04BS-109-WT induced more rapid and robust internalization in BT474, JIMT-1, and RT-112 cells, with internalization rates of the biparatopic antibodies reaching approximately 60% in JIMT-1 and RT-112 cells and up to approximately 80% in BT474 cells.

[0339] To further verify that the anti-HER2 biparatopic antibody is transported to lysosomes after internalization, this example used confocal microscopy to detect the intracellular localization of the antibody. The specific procedure was as follows: SKBR-3 cells were harvested and seeded onto five 8-well chamber slides (Thermo). After growing the cells into a uniform monolayer, the medium was discarded, the cells were rinsed once with PBS, and 500 μL of 10 μg / mL anti-HER2 biparatopic antibody 04BS-1123-ST06, a control antibody trastuzumab, or a human IgG isotype control antibody (e.g., the anti-HIV / gp120 antibody described in International Publication No. 2003106496) was added to each well of the chamber slide and incubated on ice for 1 hour. One chamber slide was used as a 0-hour control, rinsed once with ice-cold PBS, and then fixed with 2% paraformaldehyde. The other four slides were incubated at 37°C for 0.5 hours, 2 hours, 4 hours, and 8 hours, respectively, then rinsed once with ice-cold PBS and fixed overnight with 2% paraformaldehyde at 4°C. All slides were rinsed twice with PBST (PBS containing 0.05% Tween-20) and then incubated at room temperature for 10 minutes with 250 μL of fixation and permeabilization solution (PBS containing 2% goat serum and 0.5% Triton X-100). After rinsing twice with PBST, 250 μL of mouse anti-LAMP1 antibody (1:50 dilution, BD) diluted in FACS buffer (PBS containing 2% FBS) and AF488-labeled goat anti-human IgG antibody (2 μg, Molecular Probe) were added to each slide, and incubated at room temperature in the dark for 1 hour. The slides were rinsed twice with PBST and incubated in the dark at room temperature for 1 hour with 250 μL of FACS buffer containing AF647-labeled goat anti-mouse IgG antibody (1:250 dilution, Molecular Probe). After rinsing twice with PBST, DAPI was added to each slide at a final concentration of 1 μg / mL and stained for 1 minute. The slides were rinsed once with PBST, sealed with mounting medium (Thermo), and then left in the dark at room temperature overnight. Representative images obtained by scanning and analyzing with a confocal microscope are shown in Figure 12.In cells incubated with human IgG isotype control antibodies, no green fluorescence signal was detected at any time point, indicating the absence of nonspecific antibodies that bind to cells. Green fluorescence signals were detected in cells incubated with the positive control antibody trastuzumab, with no significant difference at any time point. They showed a uniform ring-shaped distribution on the cell membrane and did not overlap with red fluorescently labeled lysosomes, indicating that trastuzumab can bind to HER2 on SKBR-3 cells, but significant internalization and lysosomal transport did not occur. In contrast, in cells incubated with the anti-HER2 biparatopic antibody 04BS-1123-ST06, as treatment time progressed, the green fluorescence signal gradually shifted from the cell membrane to the intracellular compartment, overlapping with red fluorescently labeled lysosomes (appearing yellow, as indicated by the arrows in the figure). Subsequently, the intensity of the yellow fluorescence signal gradually weakened or disappeared over time, indicating that the internalized anti-HER2 biparatopic antibody 04BS-1123-ST06 was transported to and degraded by lysosomes, with a lysosomal transport efficiency of nearly 100%.

[0340] Western blotting detection of HER2 degradation further confirmed that the anti-HER2 biparatopic antibody, after binding to HER2 on the cell surface, is internalized and transported to lysosomes for degradation. The specific method is as follows: BT474 cells were collected, and 1 × 10⁶ cells were extracted. 6The cells were resuspended in complete medium (RPMI-1640 medium containing 10% FBS) to a density of cells / mL, and then dispensed into 1.5 mL EP tubes at a rate of 500 μL per tube. The test antibodies (containing biparatopic antibody 04BS-1123-ST06, control antibody trastuzumab, and monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 corresponding to the biparatopic antibody) were diluted to 300 μg / mL in complete medium, and then added to the aforementioned EP tubes at a rate of 100 μL / tube (final antibody concentration: 50 μg / mL). The EP tubes were incubated at 37°C / 5% CO2 for 24 hours. The cells were harvested by centrifugation, washed twice with ice-cold PBS, and then dissolved in 100 μL of RIPA lysis buffer (Proteintech) by incubation on ice for 30 minutes. After high-speed centrifugation at 4°C for 10 minutes, the supernatant of the cell lysate was collected, and the protein concentration in the extract was quantified using a BCA kit (Thermo). Depending on the protein concentration, each protein sample was diluted to 0.125 μg / μL with RIPA lysis buffer, and then 5× SDS-PAGE sample buffer containing DTT was added. The mixture was heated at 95°C for 5 minutes to prepare the SDS-PAGE loading sample. For electrophoresis, the loading sample was loaded onto a 10% SDS-PAGE gel at a rate of 10 μL / lane (i.e., 1 μg / lane). At the end of electrophoresis, the proteins in the gel were transferred onto a PVDF membrane at a voltage of 80 V. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour, and then cut in half along the 55 kDa molecular weight marker line. The upper half of the PVDF membrane was treated with rabbit anti-human HER2 antibody (CST) diluted 1:5000 and incubated overnight at 4°C. The lower half of the PVDF membrane was treated with mouse anti-human GAPDH antibody (Proteintech) diluted 1:10,000 and incubated at room temperature for 1 hour. At the end of incubation, the membrane was washed three times with PBST, and goat anti-rabbit IgG-HRP antibody (Jackson Immuno) and goat anti-mouse IgG-HRP antibody (Jackson Immuno) diluted 1:5000 were added to the corresponding half of the membrane and incubated at room temperature for 1 hour.The membranes were washed three times with PBST, and then exposed and developed using ECL solution (Vazyme). As shown in Figure 13, the biparatopic antibody 04BS-1123-ST06 induced almost complete degradation of HER2 in BT474 cells, while the control antibody failed to induce HER2 degradation.

[0341] In this example, the effect of the biparatopic antibody 04BS-1123-ST06 on the proliferation of BT474 cells was also tested. The specific method was as follows: BT474 cells were seeded in 96-well white-walled plates (10,000 cells / well). Test antibodies (containing 04BS-1123-ST06, trastuzumab, pertuzumab, Hu2117-HK304-06, and Hu2126-H2K1-L71-H72b-Mu14) were serially diluted and then added to the plates. The cells were incubated at 37°C / 5% CO2 for 5 days, and then the detection reagent CellCounting-Lite 2.0 Luminescent Reagent (NanoVizon) was added. Chemiluminescence values ​​were measured using a multifunctional microplate reader (Varioskan, Thermo), and the obtained data were analyzed using GraphPad Prism9 software. As shown in Figure 14, the biparatopic antibody 04BS-1123-ST06 effectively inhibited the proliferation of BT474 cells, while the corresponding monospecific antibody did not show inhibitory activity against BT474 cell proliferation. Since BT474 cell proliferation depends on signaling activation caused by homodimers formed by overexpressed HER2 on the cell membrane, it is suggested that the inhibitory activity of the biparatopic antibody against cell proliferation is due to the internalization and degradation of HER2 on the cell surface, which suppresses the formation of HER2 homodimers.

[0342] 4.5 Efficacy of anti-HER2 biparatopic antibodies against NRG-1-induced HER2 / HER4 dimerization The Promega NanoBiT structural complementarity reporter system was used to further investigate whether an anti-HER2 biparatopic antibody affects NRG-1-induced HER2 / HER4 dimerization. The NanoBiT system consists of LgBiT and SmBiT subunits, which can be fused to the two target proteins under test, respectively. When the two target proteins interact to form a dimer, the LgBiT and SmBiT subunits structurally complement each other to form a functional luciferase, which then reacts with the substrate to generate a luminescence signal. In this example, DNA sequences encoding the extracellular and transmembrane regions of HER2 and HER4 were first inserted into the NanoBiT system vectors pBiT1.3-C and pBiT2.3-C using molecular cloning techniques. These two plasmids were then co-transfected into U2OS cells. Cells were harvested 24 hours after transfection, seeded in 96-well white-walled plates, and cultured overnight at 37°C. Biparatopic 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 then mixed with NRG-1 in a 1:1 volume ratio at the time of use (the final concentration of NRG-1 was 1 nM). Nano-Glo Live Cell Reagent (Promega) was added to a 96-well white-walled plate, and the chemiluminescence value was measured as background. Subsequently, the samples prepared above were added to each well. After incubation at 37°C for 6 minutes, the chemiluminescence value was measured, and the experimental data were analyzed using GraphPad Prism9 software. As shown in Figure 15, the control antibodies trastuzumab and pertuzumab significantly inhibited NRG-1-induced HER2 / HER4 dimerization.In contrast, the chimeric antibodies mAb2117 and mAb2126, the monospecific humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (Figure 15A), and the biparatopic antibody 04BS-1123-ST06 (Figure 15B), constructed based on these two monospecific humanized antibodies, all showed no inhibitory effect. This result further indicates that anti-HER2 biparatopic antibodies do not interfere with the normal biological function of HER2 and therefore have a very low risk of inducing cardiotoxicity in vivo.

[0343] 4.6 Effect of anti-HER2 biparatopic antibodies on NRG-1-induced activation of HER2 downstream signaling pathways Human induced pluripotent stem cells (iPSCs) and related culture kits were purchased from Fujifilm Cellular Dynamics. Following the manufacturer's instructions, iPSCs were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 1 week to fully differentiate into human cardiomyocytes (iCell Cardiomyocytes). The effect of the anti-HER2 biparatopic antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes was detected using the method described in Section 1.10 of Example 1. As shown in Figure 16, unlike the positive control antibody pertuzumab, 04BS-1123-ST06 did not have an inhibitory effect on NRG-1-induced AKT phosphorylation in human cardiomyocytes, indicating that the biparatopic antibody does not interfere with NRG-1-induced HER2 dimerization and the regulation of downstream signaling pathways in cardiomyocytes.

[0344] 4.7 Modification of Fc-mediated effector function in anti-HER2 biparatopic antibodies To reduce safety risks associated with Fc-mediated effector function, three point mutations (known as TM mutations) L234F, L235E, and P331S were introduced into the Fc region of the anti-HER2 biparatopic antibody 04BS-1123-ST06 to reduce the antibody's binding activity to the Fc receptor and C1q. ForteBio analysis confirmed that the biparatopic antibody 04BS-1123-ST06 containing the TM mutations did not show significant binding to FcγRI, FcγRIIa(167H), FcγRIIb, FcγRIIIa(176V), FcγRIIIa(176F), or C1q. In contrast, the binding affinity of the biparatopic antibody to FcRn was not significantly different compared to the control antibody trastuzumab (data not shown).

[0345] Furthermore, to further verify whether the biparatopic antibody 04BS-1123-ST06 containing the TM mutation possesses ADCC activity, an ADCC assay was performed. Jurkat cells expressing FcγRIIIa-176V receptor and firefly luciferase under the control of the NFAT response element (Promega) were used as effector cells, HER2-overexpressing BT474 cells were used as target cells, and trastuzumab was used as the positive control antibody. The specific method is as follows: BT474 cells were recovered by centrifugation, resuspended in ADCC assay buffer (RPMI-1640 medium containing 0.5% FBS), and 1.5 × 10⁶ cells were placed on a 96-well white-wall plate. 4 The test antibody was added at a density of cells / well. The test antibody was serially diluted with ADCC assay buffer and added to a 96-well plate at a rate of 25 μL / well. Effector cells were collected by centrifugation, resuspended in ADCC assay buffer, and 1.5 × 10⁶ cells were added. 5Cells were added to the aforementioned 96-well plates at the cell / well density, and then incubated overnight at 37°C and 5% CO2. The following day, the 96-well plates were equilibrated to room temperature, and then the detection reagent Bio-Lite luciferase reagent (Vazyme, Nanjing) was added. Chemiluminescence values ​​were measured using a multifunctional microplate reader (Varioskan, Thermo), and the obtained data were analyzed using GraphPad Prism9 software. As shown in Figure 17, unlike the positive control antibody trastuzumab, 04BS-1123-ST06 did not induce luciferase expression even at the highest concentration, indicating that the anti-HER2 biparatopic antibody with the TM mutation introduced into the Fc region does not possess ADCC activity.

[0346] Example 5: Preparation and Characterization of Functional Activity of Anti-HER2 Biparatopic ADCs 5.1 Preparation of anti-HER2 biparatopic antibody-drug conjugates This example describes the preparation of an anti-HER2 biparatopic ADC by conjugation of the anti-HER2 biparatopic antibody 04BS-1123-ST06 to eribulin, a cytotoxic small molecule compound with antitumor activity, via a cleavable linker (e.g., Mal-(PEG)2-VC-PAB or Mal-(PEG)2-GGFG). Eribulin was prepared according to the method described in ZL201910197071.8 or ZL201910509222.9 and International Publication No. 1999065894.

[0347] The payload Mal-(PEG)2-VC-PAB-eribulin was prepared according to the method described in Example 2.1.1 of International Publication No. 2017151979.

[0348] The preparation method for the payload Mal-(PEG)2-GGFG-eribulin is as follows: NH2-(PEG)2-COOH and maleic anhydride were mixed in acetic acid and heated overnight under reflux, after which the acetic acid was removed by rotary evaporation. The residue was purified by HPLC and freeze-dried to obtain Mal-(PEG)2-COOH. Using 2Cl Trt Resin as the solid support, the Fmoc protecting group was removed with 20% piperidine / DMF (v / v). Using HOBT / DIC as the condensation system and DMF as the reaction solvent, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Gly-OH were sequentially linked. The product was then cleaved in a solution of DCM:TFE:AcOH=7:2:1, precipitated with methyl tert-butyl ether, centrifuged, dried, and further purified by HPLC to obtain Fmoc-GGFG-OH. Fmoc-GGFG-OH and eribulin were placed in a necked flask, dissolved in DMF, and cooled to 0°C. DIPEA (161.3 mg, 1.25 mmol) and DECP (122.3 mg, 0.75 mmol) were added, and the reaction was carried out 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 was washed with MTBE to obtain crude Fmoc-GGFG-eribulin. Crude Fmoc-GGFG-eribulin was placed in a necked flask, THF was added, and cooled to 0°C. Two equivalents of LiOH aqueous solution were added, and the reaction was carried out at room temperature and monitored by HPLC. After completion, the pH was adjusted to 6-7 with 50% acetic acid. THF was removed by rotary evaporation, and the product was purified by HPLC to obtain pure NH2-GGFG-eribulin. HATU was added at room temperature to a DMF solution containing Mal-(PEG)2-COOH and sodium bicarbonate. After stirring for 30 minutes, an equal volume of free NH2-GGFG-eribulin was added to the solution, and the mixture was stirred for another hour until the reaction was complete, as determined by TLC. The solid was filtered off, and the filtrate was purified by HPLC to obtain Mal-(PEG)2-GGFG-eribulin.

[0349] The preparation method for anti-HER2 biparatopic ADCs was as follows: ZnCl2 (3.0 mM) and TCEP (6.0 mM) were slowly added to the anti-HER2 biparatopic antibody 04BS-1123-ST06 (prepared in 1.5 mM, 50 mM histidine-HCl, 8% sucrose, pH 5.5) in an ice bath. The final reaction concentrations of ZnCl2, TCEP, and 04BS-1123-ST06 antibody were 0.10 mM, 0.20 mM, and 0.05 mM, respectively. After mixing, the mixture was allowed to react 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 in an ice bath to a final concentration of 0.32 mM. After continuing the reaction at 8°C for 3 hours, cysteine ​​was added to a final concentration of 0.05 mM to remove excess Mal-(PEG)2-VC-PAB-eribulin or Mal-(PEG)2-GGFG-eribulin. Then, EDTA was added to a final concentration of 0.15 mM to remove Zn 2+ The related impurities were chelated. 2 The residues were removed using [a specific method]. Finally, the drug-to-antibody ratio (DAR) and heterogeneity of the prepared ADCs were analyzed by HIC-HPLC (BioCore HIC-butyl 5 μm / 4.6 × 100 mM, purchased from NanoChrom), and the purity of the ADC products was analyzed by SEC-HPLC (Zenix-C SEC-300, purchased from Sepax Technologies).

[0350] The average DAR value of the prepared ADC can be calculated using the following formula: Average DAR = [AUC +1 +2(AUC +2 ) + 3 (AUC +3 )+...+n(AUC +n )] / ΣAUC 合計 ] where AUC +1 AUC refers to the area under the curve of the peak corresponding to an ADC bound to a single small molecule compound. +2ΣAUC refers to the area under the curve of the peak corresponding to the ADC bound to the two small molecule compounds, and so on. 合計 This is the sum of the areas under the curve of all peaks. The average DAR value of the ADCs prepared in this example was 4.0 ± 0.5, and the purity was >95%.

[0351] 5.2 Verification of antigen-binding specificity of anti-HER2 biparatopic ADCs The binding activity of an exemplary anti-HER2 biparatopic ADC (ST06-VCP-eribulin) to recombinant HER2 protein and its cross-reactivity to other members of the human ErbB / HER family were verified by ELISA according to the method described in Section 1.4 of Example 1, with antigens including recombinant proteins of the extracellular domains of human HER2, EGFR, HER3, and HER4. As shown in Figure 18, the anti-HER2 biparatopic ADC (ST06-VCP-eribulin) was able to specifically bind to HER2 and did not exhibit cross-reactivity to other members of the human ErbB / HER family. The antigen-binding specificity of the anti-HER2 biparatopic ADC was identical to that of its corresponding unbound antibody 04BS-1123-ST06 (data not shown), indicating that conjugation with a small molecule toxin compound did not alter the antigen-binding specificity of the biparatopic antibody.

[0352] 5.3 Detection of in vitro tumor cell-killing activity of anti-HER2 biparatopic ADCs In this example, the in vitro cell-killing activity of anti-HER2 biparatopic ADCs was evaluated using a panel of tumor cell lines expressing various levels of HER2. Table 13 shows the HER2 expression levels and drug resistance status of the tumor cell lines used in the assay. Here, JIMT-1 is a cell line that overexpresses HER2 and exhibits intrinsic resistance (in vitro activity) to DS-8201, while SKOV-3R* is a cell line that overexpresses HER2 and exhibits acquired resistance to DS-8201.

[0353] The procedure for investigating in vitro tumor cell killing activity was as follows: Cells were harvested by trypsin treatment, resuspended in a medium containing 10% FBS, and then seeded in 96-well white-walled plates at a density of 5,000 or 10,000 cells / well. Cells were cultured in a 37°C incubator containing 5% CO2. After the cells had fully adhered, ST06-VCP-eribulin, ST06-GGFG-eribulin, or DS-8201, serially diluted in the corresponding culture medium, was added to the wells. Depending on the growth rate of each cell line, the plates were incubated at 37°C and 5% CO2 for 3–5 days. At the end of incubation, the cell culture plates were removed from the incubator, equilibrated to room temperature, CellCount Lite 2.0 reagent (Vazyme, Nanjing) was added, and the plates were shaken for 2–5 minutes to completely lyse the cells. After incubation at room temperature for 10 minutes, chemiluminescence values ​​were measured using a multi-functional microplate reader (Varioskan, Thermo). Data were analyzed using GraphPad Prism9 software, and the results were expressed as a percentage of the chemiluminescence value relative to the untreated control well.

[0354] The results are summarized in Figure 19 and Table 13. In HER2-overexpressing tumor cell lines (NCI-N87, SKBR-3, and SKOV-3), anti-HER2 biparatopic ADCs (including ST06-GGFG-eribulin and ST06-VCP-eribulin) and the benchmark molecule DS-8201 showed potent cytotoxicity, with biparatopic ADCs exhibiting significantly higher cytotoxic activity than DS-8201. In HER2-low-expressing tumor cell lines (MDA-MB-361 and MDA-MB-453), the cytotoxic activity of biparatopic ADCs was significantly higher than that of DS-8201. In the HER2-low-expressing cell line ZR-75-1, biparatopic ADCs showed effective cytotoxic activity, while DS-8201 showed little to no cytotoxic effect. Furthermore, in tumor cell lines expressing even lower levels of HER2 (T47D, RT-112, MCF-7) and the HER2-null tumor cell line MDA-MB-468, biparatopic ADCs did not show significant cytotoxicity, demonstrating a favorable safety margin for biparatopic ADCs. In addition, in JIMT-1 cells, a cell line that overexpresses HER2 and has innate / endogenous resistance to DS-8201, biparatopic ADCs (including ST06-GGFG-eribulin and ST06-VCP-eribulin) also showed significant cytotoxicity. Therefore, anti-HER2 biparatopic ADCs have a broader range of cytotoxic activity, capable of killing not only HER2-overexpressing tumor cells but also tumor cells with low HER2 expression, and even tumor cells that do not respond to DS-8201.

[0355] To further investigate whether anti-HER2 biparatopic ADCs can kill tumor cells that have developed resistance or tumor cells that are relapsed / refractory to DS-8201 treatment, this example isolated tumor cell lines (named SKOV-3R*) that had acquired resistance to DS-8201 by maintaining and passaging HER2-overexpressing SKOV-3 cells in culture medium containing 100 nM DS-8201, with the concentration of DS-8201 gradually increasing until the cells could grow at a normal rate. In vitro cytotoxicity assays showed that biparatopic ADCs (including ST06-GGFG-eribulin and ST06-VCP-eribulin) could effectively kill SKOV-3R* cells that had acquired drug resistance.

[0356] [Table 13]

[0357] 5.4 Bystander killing activity of anti-HER2 biparatopic ADCs The bystander-killing activity of the anti-HER2 biparatopic ADC ST06-GGFG-eribulin was evaluated using the HER2-overexpressing cell line BT474 and the HER2-null cell line MDA-MB-468. The specific method was as follows: On day 0, BT474 cells were seeded at 10,000 cells / 100 μL / well in a 96-well white-walled plate and incubated overnight at 37°C. On day 1, ST06-GGFG-eribulin was diluted to 20 nM in medium, then serially diluted 5-fold and added to the plate at 100 μL / well (i.e., a starting concentration of 10 nM). The plate was incubated at 37°C. On day 3, MDA-MB-468 cells were seeded at 10,000 cells / well in a new 96-well white-walled plate and incubated overnight at 37°C. On day 4, the MDA-MB-468 cell culture medium was removed and replaced with 100 μL / well of conditional medium recovered from the BT474 cell culture plate to evaluate the bystander effect of ADC. Simultaneously, freshly prepared ST06-GGFG-eribulin was added to the MDA-MB-468 cells at 100 μL / well as a control, in 5-fold serial dilutions starting from 10 nM. Furthermore, the BT474 cell cultures from day 4 onward were assayed for cell viability using CellCount Lite 2.0 reagent. Plates containing MDA-MB-468 cells were incubated at 37°C for 72 hours, and cell viability was detected by adding CellCount Lite 2.0 reagent. The obtained data were analyzed using GraphPad Prism 9 software. As shown in Figure 20A, ST06-GGFG-eribulin caused significant cytotoxicity in BT474 cells after 3 days of treatment, and MDA-MB-468 cells cultured with BT474 condition medium also showed significant cell death. However, the newly prepared ADC solution did not exhibit cytotoxic activity against MDA-MB-468 cells. These results suggest that while ST06-GGFG-eribulin kills BT474 cells, the small molecule toxin compound eribulin released into the BT474 cell culture medium can kill HER2 null MDA-MB-468 cells.

[0358] Furthermore, the bystander-killing activity of ST06-GGFG-eribulin was further investigated in this example by co-culturing BT474 cells and HER2 null Jurkat cells. The specific method is as follows: BT474 cells and GFP-expressing Jurkat cells were collected, and both cell types were placed in a 24-well cell culture plate in 1 × 10⁶ wells. 5 Cells were seeded per well, in separate wells or in the same well. ST06-GGFG-eribulin was added to a final concentration of 1 nM. After incubation at 37°C for 72 hours, cells from each well were collected into flow cytometry tubes. After centrifugation and supernatant removal, cells were resuspended in 100 μL of FACS buffer (PBS containing 1% BSA), 3 μL of 7-AAD was added for dead cell staining, and then flow cytometry analysis was performed. For each tube, 10 μL of cell suspension was collected and analyzed. After excluding 7-AAD-positive dead cells, BT474 and Jurkat cells were distinguished using lateral scattering channels and GFP channels. Finally, the bystander activity of biparatopic ADCs was assessed by comparing the number of viable cells after treatment with either ST06-GGFG-eribulin or blank culture medium (i.e., the control group referred to as "medium"). As shown in Figure 20B, ST06-GGFG-eribulin showed significant cytotoxic activity against BT474 cells, but did not show cytotoxicity against Jurkat cells in monoculture. However, ST06-GGFG-eribulin significantly killed both BT474 and Jurkat cells in co-cultures of the two cell types.

[0359] In summary, the results of both experiments demonstrate that ST06-GGFG-eribulin exhibits bystander-killing activity.

[0360] Example 6: Evaluation of antitumor activity of anti-HER2 biparatopic ADC in a mouse subcutaneous xenograft model. 6.1 Antitumor activity of anti-HER2 biparatopic ADCs in a mouse subcutaneous xenograft model established using tumor cell lines In this example, the antitumor activity of anti-HER2 biparatopic ADCs was evaluated in three mouse subcutaneous xenograft tumor models established using human tumor cell lines, with DS-8201 (brand name Enhertu, purchased from Daiichi Sankyo) used as a reference drug. The three tumor models were as follows: HER2-overexpressing NCI-N87 gastric cancer model, HER2-moderately expressing JIMT-1 breast cancer model, and HER2-low expressing RT-112 bladder cancer model. The specific method was as follows: cell lines (NCI-N87, JIMT-1, and RT-112) were cultured until the logarithmic growth phase, then cells were harvested and 5-10 × 10⁶ cells were collected per mouse. 6 The cells were subcutaneously inoculated into the dorsal side of the right forelimb of immunodeficient mice (Nude or SCID) at a given cell density. The tumor measured 200 ± 50 mm. 3 Once the tumor volume reached a certain level, the mice were randomized into groups including the ST06-GGFG-eribulin test group, the DS-8201 reference control group, the vehicle control group (50 mM histidine-HCl solution containing 8% sucrose, pH 5.5), and the antibody-small molecule drug mixture ADMix control group (ADMix was prepared by mixing unbound antibody 04BS-1123-ST06 and eribulin in a molar ratio of 1:4) (8 tumor-bearing mice per group). All tumor-bearing mice were administered intravenously. The doses and administration frequencies for each group in different models are shown in Figure 21. After administration, the body weight and tumor dimensions (length and width) of the mice were measured and recorded twice a week. Tumor volume was calculated using the following formula: Tumor volume = (length) × (width) 2 ×0.5.

[0361] As shown in Figure 21, both DS-8201 and ST06-GGFG-eribulin showed significant tumor growth inhibitory activity in the NCI-N87 (Figure 21A), JIMT-1 (Figure 21B), and RT-112 (Figure 21C) tumor models compared to vehicle controls. Furthermore, the antitumor activity of ST06-GGFG-eribulin showed a dose-dependent trend in both the NCI-N87 and JIMT-1 models. At the same dose level (3 mg / kg), ST06-GGFG-eribulin showed antitumor activity comparable to DS-8201 in the HER2-overexpressing NCI-N87 model, but significantly stronger than DS-8201 in the HER2-moderately expressing JIMT-1 model (p<0.01). In the HER2-low-expression RT-112 model, considering the differences in molecular weight and DAR values, ST06-GGFG-eribulin demonstrated significantly superior antitumor activity compared to DS-8201 when administered at a DAR-equivalent dose (9 mg / kg), and the difference in antitumor activity was statistically significant (p<0.01). Throughout the study, no weight loss or other adverse toxic side effects were observed in tumor-bearing mice (data not shown).

[0362] 6.2 Antitumor activity of anti-HER2 biparatopic ADCs in a mouse subcutaneous xenograft tumor model with acquired resistance to DS-8201 To further investigate whether anti-HER2 biparatopic ADCs can kill tumors that have acquired resistance to or relapsed in response to DS-8201 treatment, this example established a mouse xenograft tumor model that had acquired resistance to DS-8201 using the NCI-N87 tumor cell line. The method was as follows: Wild-type NCI-N87 cells were maintained and subcultured in a medium containing gradually increasing concentrations of DS-8201. A subpopulation of cell lines with certain resistance to DS-8201 was isolated and named NCI-N87(R). NCI-N87(R) cells were grown and cultured in RPMI-1640 medium containing 10% serum until they reached the exponential growth phase, then harvested and resuspended in a mixture of PBS and Matrigel (volume ratio 1:1). 1 × 10⁶ cells were placed on the dorsal side of the right forelimb of BALB / c nude mice. 7Cells / mice were subcutaneously inoculated, with 5 female mice inoculated at each time point. In vivo screening for tumors with acquired drug resistance was performed as follows: tumor volume of 200 ± 50 mM. 3 When the tumor reached a certain stage, DS-8201 (3 mg / kg) was administered intravenously once a week. Tumor-bearing mice whose tumors continued to grow stably after administration were screened, with the number of dose administrations ranging from 3 to 5 depending on the tumor growth rate. 3 Once the tumor reached a certain size, the mouse was euthanized and the tumor was harvested. After removing the necrotic tissue, the tumor was measured to approximately 30 mm. 3 The tumor was cut into small pieces and subcutaneously inoculated dorsally near the right forelimb of five new female nude mice for a subsequent series of DS-8201 treatments and screenings. After three screenings, the tumors in all five tumor-bearing mice showed stable growth after administration of DS-8201 (3 mg / kg dose), and the growth rate was relatively consistent. The tumors reached approximately 1000 mm². 3 Once it reaches this stage, two mice are euthanized, the tumors are harvested, and after removing the necrotic tissue, the tumors are reduced to approximately 30 mm. 3 The sample was cut into halves and subcutaneously inoculated into the dorsal side near the right forelimb of 50 female nude mice.

[0363] From these 50 tumor-bearing mice, relatively uniform tumor growth and approximately 210 mm were observed. 3Twenty-nine mice with an average tumor volume were selected and randomized into groups including two ST06-GGFG-eribulin treatment groups (3 mg / kg dose group and 10 mg / kg dose group), one DS-8201 control group (3 mg / kg dose), and one vehicle control group. The ST06-GGFG-eribulin 3 mg / kg treatment group, DS-8201 control group, and vehicle control group each consisted of eight mice, while the ST06-GGFG-eribulin 10 mg / kg treatment group consisted of five mice. The mice in each group were administered intravenously for a total of four weeks. The DS-8201 and vehicle groups received the drug once a week, while the ST06-GGFG-eribulin treatment groups received it once in the first week, and then twice a week for the remaining three weeks. The body weight and tumor size of the mice were measured and recorded twice a week for each group. The tumor volume was calculated using the following formula: Tumor volume = (length) × (width) 2 ×0.5.

[0364] As shown in Figure 22, mouse tumors showed continued and stable growth after DS-8201 treatment, demonstrating the successful establishment of the model, i.e., the tumor model was confirmed to be a xenograft model with acquired resistance to DS-8201. ST06-GGFG-eribulin showed antitumor activity at both 3 mg / kg and 10 mg / kg doses, and in particular, at the 10 mg / kg dose (equivalent to the DAR of DS-8201), ST06-GGFG-eribulin induced almost complete tumor regression, indicating that the biparatopic ADC (ST06-GGFG-eribulin) was able to overcome acquired resistance to DS-8201. These results suggest the therapeutic potential of biparatopic ADCs in the treatment of relapsed / refractory tumors from DS-8201 therapy. No weight loss or other adverse toxic side effects were observed in tumor-bearing mice during the study (data not shown).

Claims

1. An isolated anti-HER2 antibody or its antigen-binding fragment, characterized by the following: (1) The anti-HER2 antibody or its antigen-binding fragment can specifically bind to subdomains 1, 3 and / or 4 of the HER2 extracellular domain. (2) The anti-HER2 antibody or its antigen-binding fragment does not affect HER2 and the downstream signaling pathways mediated by it, and does not induce, block or inhibit phosphorylation and / or dephosphorylation of HER2 tyrosine residues, and / or does not induce, block or inhibit ligand-dependent or ligand-independent HER2 dimerization and its downstream signaling pathways, wherein the HER2 dimerization includes HER2 / HER4 dimerization and HER2 / HER3 dimerization, and the ligand includes NRG-1 and heregulin, (3) The anti-HER2 antibody or its antigen-binding fragment does not cross-react with other members of the human ErbB / HER family (including EGFR, HER3, and HER4). An anti-HER2 antibody or its antigen-binding fragment containing at least one of the following.

2. The anti-HER2 antibody or its antigen-binding fragment can specifically bind to subdomain 3 of the HER2 extracellular region, and (1) X １４ HCDR1 having the amino acid sequence of YGMS (SEQ ID NO: 217) (where X １ = S, N or D), (2) SISGX ２ GX ３ YX ４ KYXX ５ X ６ X ７ HCDR2 having the amino acid sequence of VKXG (SEQ ID NO: 218) (where X ２ = G or S, X ３ = S or N, X ４ = T or A, X ５ = P, A, G or V, X ６ = D, G, E, P, Q or R, X ７ = S, K or N), (3) HCDR3 having the amino acid sequence of DYXFFDV (SEQ ID NO: 219) (where X ８ = A, I, N, R, S or V), (4) LCDR1 having the amino acid sequence of RSSQSLXXSNXTYLH (SEQ ID NO: 220) (where X ８ = A, I, N, R, S or V), (4) RSSQSLXXSNXTYLH (SEQ ID NO: 220) (where X ９ X １０ SNX １１ = V or L, X ９ = H or S, X １０ = G, A, I, S, R or T), (5) LCDR2 having the amino acid sequence of KVSNRXS (SEQ ID NO: 221) (where X １１ = F, D or P), (6) LCDR3 having the amino acid sequence of XQSTHVPT (SEQ ID NO: 222) (where X １２ = S or Q, X １２ = F, D or P), (6) XQSTHVPT (SEQ ID NO: 222) (where X １３ = S or Q, X １４ = Y or W), or the anti-HER2 antibody or its antigen-binding fragment according to claim 1, comprising 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 the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively.​​​​

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

4. The above anti-HER2 antibody or its antigen-binding fragment can specifically bind to subdomain 1 of the HER2 extracellular region, (1) DYX １５ HCDR1 (wherein X) has the amino acid sequence of MH (SEQ ID NO: 223) １５ (1) S or A), (2) WINTX １６ TGX １７ PTYADX １８ X １９ HCDR2 having the amino acid sequence of KG (SEQ ID NO: 224) (wherein 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) VGX ２０ X ２１ X ２２ HCDR3 having the amino acid sequence of YAMDY (SEQ ID NO: 225) (wherein X ２０ = R or Y, X ２１ = Y or G, X ２２ = D or S), (4) X ２３ LCDR1 (wherein X) has the amino acid sequence ASQDVYTAVA (SEQ ID NO: 226) ２３ (5) X ２４ ASX ２５ RX ２６ LCDR2 having the amino acid sequence of T (SEQ ID NO: 227) (wherein 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 (6) QQX ２７ LCDR3 (wherein X) has the amino acid sequence YSTPPT (SEQ ID NO: 228) ２７ An anti-HER2 antibody or antigen-binding fragment thereof according to claim 1, comprising 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 with respect to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

5. Including HCDR1 shown in sequence number 98 or 99, HCDR2 shown in sequence numbers 100, 101, 102, 103, 104, 105, 106, 107, 108 or 109, HCDR3 shown in sequence numbers 110, 111, 112 or 113; and LCDR1 shown in sequence number 114 or 115, LCDR2 shown in sequence numbers 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138, and LCDR3 shown in sequence numbers 139, 140, 141 or 142, moreover, (1) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID No. 100, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 114, LCDR2 shown in Sequence ID Nos. 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128, LCDR3 shown in Sequence ID No. 139, or (2) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID No. 100, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 114, LCDR2 shown in Sequence ID No. 116, LCDR3 shown in Sequence ID No. 140, 141 or 142, or (3) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID Nos. 101, 102, 103, 104, 105, 106, 107, 108 or 109, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 114, LCDR2 shown in Sequence ID No. 116, LCDR3 shown in Sequence ID No. 139, or (4) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID No. 100, HCDR3 shown in Sequence ID No. 111, 112 or 113, LCDR1 shown in Sequence ID No. 114, LCDR2 shown in Sequence ID No. 116, LCDR3 shown in Sequence ID No. 139, or (5) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID No. 100, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 115, LCDR2 shown in Sequence ID No. 116, LCDR3 shown in Sequence ID No. 139, or (6) HCDR1 shown in Sequence ID No. 99, HCDR2 shown in Sequence ID No. 100, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 114, LCDR2 shown in Sequence ID No. 116, LCDR3 shown in Sequence ID No. 139, or (7) HCDR1 shown in Sequence ID No. 98, HCDR2 shown in Sequence ID No. 102, HCDR3 shown in Sequence ID No. 110, LCDR1 shown in Sequence ID No. 115, LCDR2 shown in Sequence ID Nos. 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138, LCDR3 shown in Sequence ID No. 142 Includes, Preferably, an anti-HER2 antibody or antigen-binding fragment thereof according to claim 1 or 4, comprising HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 102, HCDR3 shown in SEQ ID NO: 110, LCDR1 shown in SEQ ID NO: 115, LCDR2 shown in SEQ ID NO: 129, 133, 136 or 138, and LCDR3 shown in SEQ ID NO:

142.

6. The above anti-HER2 antibody or its antigen-binding fragment can specifically bind to subdomain 4 of the HER2 extracellular region, and may include HCDR1 shown in SEQ ID NO: 199, HCDR2 shown in SEQ ID NO: 200, HCDR3 shown in SEQ ID NO: 201, LCDR1 shown in SEQ ID NO: 202, LCDR2 shown in SEQ ID NO: 203, LCDR3 shown in SEQ ID NO: 204, or The above anti-HER2 antibody or its antigen-binding fragment can specifically bind to subdomain 1 of the HER2 extracellular region, and may include HCDR1 shown in SEQ ID NO: 205, HCDR2 shown in SEQ ID NO: 206, HCDR3 shown in SEQ ID NO: 207, LCDR1 shown in SEQ ID NO: 208, LCDR2 shown in SEQ ID NO: 209, LCDR3 shown in SEQ ID NO: 210, or 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 subdomain 1 of the HER2 extracellular region, and comprises HCDR1 shown in SEQ ID NO: 211, HCDR2 shown in SEQ ID NO: 212, HCDR3 shown in SEQ ID NO: 213, LCDR1 shown in SEQ ID NO: 214, LCDR2 shown in SEQ ID NO: 215, and LCDR3 shown in SEQ ID NO:

216.

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

8. A heavy chain variable region VH having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 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 It includes a light chain variable region VL having at least 80% identity with the amino acid sequence shown in column numbers 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, moreover, (1) 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 with VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4, or (2) 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 with VH shown in SEQ ID NO: 143 and VL shown in SEQ ID NOs: 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188, respectively, or (3) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NOs. 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 VL shown in SEQ ID NOs. 171, respectively, or (4) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. Includes, Preferably, the amino acid sequence contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NOs: 169, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively. More preferably, an anti-HER2 antibody or antigen-binding fragment thereof according to claim 1, 4, or 5, comprising 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 with VH shown in SEQ ID NO: 169 and VL shown in SEQ ID NO: 189, 193, 196, or 198, respectively.

9. It includes a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are (1) 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 with VH shown in SEQ ID NO: 5 and VL shown in SEQ ID NO: 6, respectively, or (2) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO: 7 and VL shown in SEQ ID NO: 8, respectively, or (3) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO: 9 and VL shown in SEQ ID NO: 10, respectively. An anti-HER2 antibody or antigen-binding fragment thereof according to claim 1 or 6, comprising the above.

10. An anti-HER2 biparatopic antibody comprising two antigen-binding domains capable of non-competitively and simultaneously binding to the extracellular domain of HER2, wherein the first antigen-binding domain and the second antigen-binding domain each specifically bind to different epitopes on the extracellular domain of HER2, the epitopes being located in subdomains 1, 3, and 4 of the extracellular domain of HER2, and the biparatopic antibody having the following characteristics: (1) The above-mentioned biparatopic antibody can crosslink / cluster HER2 on the surface of tumor cells, thereby inducing rapid and robust internalization of the crosslinked HER2 complex, as evidenced by the internalization rates of over 60% in HER2-low-expressing tumor cells and over 80% in HER2-over-expressing tumor cells. (2) The above-mentioned biparatopic antibody effectively induces lysosomal transport and degradation of HER2 on the surface of tumor cells, significantly reducing HER2 expression on the cell surface, thereby significantly inhibiting the proliferation of HER2-overexpressing tumor cells. (3) The above-mentioned biparatopic antibody does not affect HER2 and the downstream signaling pathways it mediated, including not inducing, blocking, or inhibiting ligand-induced HER2 dimerization and the activation of its downstream signaling pathways, and therefore the above-mentioned biparatopic antibody does not interfere with the function and regulation of HER2 and the downstream signaling pathways it mediated in normal tissues / cells, including cardiomyocytes, and the above-mentioned ligand contains NRG-1. (4) The above-mentioned biparatopic antibody can specifically bind to HER2 without cross-reacting with other members of the human ErbB / HER family (including EGFR, HER3, and HER4). An anti-HER2 biparatopic antibody containing at least one of the following.

11. In the first antigen-binding domain and the second antigen-binding domain described above, one specifically binds to subdomain 1 of the HER2 extracellular region, and the other specifically binds to subdomain 3 of the HER2 extracellular region. Furthermore, the first antigen-binding domain described above is (1) X １ HCDR1 having the amino acid sequence of YGMS (wherein X １ (1) S, N or D), (2) SISGX ２ GX ３ YX ４ KYX ５ X ６ X ７ HCDR2 having the amino acid sequence of VKG (wherein X ２ = G or S, X ３ = S or N, X ４ = T or A, X ５ = P, A, G or V, X ６ = D, G, E, P, Q or R, X ７ (3) DYX ８ HCDR3 having the amino acid sequence of GFFDV (wherein X ８ = A, I, N, R, S or V), (4) RSSQSLX ９ X １０ SNX １１ LCDR1 having the amino acid sequence of NTYLH (wherein X ９ = V or L, X １０ = H or S, X １１ = G, A, I, S, R or T), (5) KVSNRX １２ LCDR2 having the amino acid sequence of S (wherein X １２ = F, D or P), (6) X １３ QSTHVPX １４ LCDR3 having the amino acid sequence of T (wherein X １３ = S or Q, X １４ =Y or W), 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 with respect to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, The second antigen-binding domain is (1) an HCDR1 having the amino acid sequence of DYX １５ MH (where X １５ = S or A), (2) an HCDR2 having the amino acid sequence of WINTX １６ TGX １７ PTYADX １８ X １９ KG (where 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) an HCDR3 having the amino acid sequence of VGX ２０ X ２１ X ２２ YAMDY (where X ２０ = R or Y, X ２１ = Y or G, X ２２ = D or S), (4) an LCDR1 having the amino acid sequence of X ２３ ASQDVTAV A (where X ２３ = K or R), (5) an LCDR2 having the amino acid sequence of X ２４ ASX ２５ RX ２６ T (where 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) an LCDR3 having the amino acid sequence of QQX ２７ YSTPPT (where X ２７ = H, S, A or Y), or the biparatopic antibody according to claim 10, comprising amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

12. The first antigen-binding domain includes HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NOs: 21, 23, 24, 27, 28, 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, and the second antigen-binding domain includes HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 102, HCDR3 shown in SEQ ID NO: 110, LCDR1 shown in SEQ ID NO: 115, LCDR2 shown in SEQ ID NOs: 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138, and LCDR3 shown in SEQ ID NO:

142. Furthermore, the anti-HER2 biparatopic antibody according to claim 10 or 11, wherein the first antigen-binding domain includes HCDR1 shown in SEQ ID NO: 11, HCDR2 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, and the second antigen-binding domain includes HCDR1 shown in SEQ ID NO: 98, HCDR2 shown in SEQ ID NO: 102, HCDR3 shown in SEQ ID NO: 110, LCDR1 shown in SEQ ID NO: 115, LCDR2 shown in SEQ ID NO: 133, and LCDR3 shown in SEQ ID NO:

142.

13. The first antigen-binding domain described above includes 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 with the VH and VL, respectively, and the second antigen-binding domain described above includes an amino acid sequence described above. The combined domain contains an amino acid sequence that is identical to the VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above VH and VL, respectively. Preferably, the first antigen-binding domain comprises 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 with the above VH and VL, respectively, and the second antigen-binding domain comprises 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 with the above VH and VL, respectively, according to claim 10 or 11.

14. Having the DVD-Ig format, in the first antigen-binding domain and the second antigen-binding domain, one is an Fv domain and the other is a Fab or IgG domain, and the Fv domain is linked to the Fab or IgG domain via a linker. Furthermore, the anti-HER2 biparatopic antibody according to any one of claims 10 to 12, wherein the first antigen-binding domain is a Fv domain that specifically binds to subdomain 3 of the HER2 extracellular region, the second antigen-binding domain is a Fab or IgG domain that specifically binds to subdomain 1 of the HER2 extracellular region, and 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 of the Fv domain is fused to the N-terminus of the VH domain of the Fab or IgG domain via a linker, and the C-terminus of the VL domain of the Fv domain is fused to the N-terminus of the VL domain of the Fab or IgG domain via a linker.

15. It contains four polypeptide chains, (1) Two of the polypeptide chains include VH1-L1-VH2-C-(Fc)n, where VH1 represents the heavy chain variable region of the first antigen-binding domain that specifically binds to subdomain 3 of the HER2 extracellular domain, L1 represents the linker, VH2 represents the heavy chain variable region of the second antigen-binding domain that specifically binds to subdomain 1 of the HER2 extracellular domain, C represents the heavy chain constant region CH1, Fc represents the heavy chain constant region Fc domain, and n is 0 or 1. (2) The other two polypeptide chains comprise VL1-L2-VL2-CL, where VL1 represents the light chain variable region of the first antigen-binding domain that specifically binds to subdomain 3 of the HER2 extracellular domain, L2 represents the linker, VL2 represents the light chain variable region of the second antigen-binding domain that specifically binds to subdomain 1 of the HER2 extracellular domain, and CL is the constant region of the IgG light chain. The above L1 and L2 linkers, (G ４ S) ｎ The array includes, in the formula, n is G ４ The anti-HER2 biparatopic antibody according to any one of claims 10 to 14, wherein the copy number n of S is an integer greater than 0, and the copy numbers n of the L1 and L2 linkers may be the same or different.

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

142.

17. The above VH1 includes 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 with the VH shown in SEQ ID NO: 73, 78, 79, or 80, and the above VH2 includes 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 with the VH shown in SEQ ID NO: 169, and the above VL1 is a sequence The anti-HER2 biparatopic antibody according to claim 15 or 16, comprising 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 with respect to VL shown in SEQ ID NO: 193, 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 with respect to VL shown in SEQ ID NO:

193.

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

233.

19. An anti-HER2 biparatopic antibody-drug conjugate (ADC) comprising an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, a small molecule toxin compound, and a cleavable linker, wherein the ADC has the following characteristics: (1) The ADC binds to two different epitopes of HER2 on the surface of tumor cells, crosslinks HER2 into clusters, induces rapid internalization and lysosomal transport of the ADC, thereby significantly improving the release of small molecule toxin compounds in the ADC to target cells. This allows the ADC to obtain a broader spectrum of cytotoxic activity, not only having stronger cytotoxic activity than existing HER2-targeting drugs in HER2-overexpressing tumor cells, but also being able to have a direct cytotoxic effect in HER2-low-expressing tumor cells. The existing HER2-targeting drugs include trastuzumab, pertuzumab, T-DM1, and DS-8201. (2) The above ADC exhibits cytotoxic effects against tumors that have acquired resistance to or have relapsed to treatment with existing HER2-targeting drugs. (3) The above ADC does not affect the biological activity of HER2 and the modulation of the signaling pathways mediated by it, does not interfere with the normal function of HER2 in cardiomyocytes, lacks Fc effector function, and thereby exhibits significantly improved safety compared to existing HER2-targeting drugs. Having at least one of the following, Furthermore, the above ADC is expressed by equation (I): Ab-(L-D) ｐ (I) In the formula, Ab represents an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain is an Fv domain that specifically binds to subdomain 3 of the HER2 extracellular region, the second antigen-binding domain is an IgG domain that specifically binds to subdomain 1 of the HER2 extracellular region, 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 severable linker. p is in the range of 2 to 8. Anti-HER2 biparatopic antibody-drug conjugate (ADC).

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

142.

21. The ADC according to claim 19 or 20, wherein the first antigen-binding domain includes 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 with the above VH and VL, and the second antigen-binding domain includes 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 with the above VH and VL.

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

233.

23. The above small molecule toxin compounds include eribulin, auristatin derivatives, tubulicin, cryptomycin, maytansinoid derivatives, topoisomerase inhibitors, pyrrolobenzodiazepines (PBD), calicheamycin and its derivatives, duocalmycin, alkylating chemotherapeutic agents and other compounds having alkylated forms, antimetabolites, mitotic inhibitors, and cytotoxic antibiotics. Furthermore, the ADC according to any one of claims 19 to 22, wherein the auristatin derivative comprises MMAE, MMAF, and MMAD; the maytansinoid derivative comprises DM1, DM2, DM3, and DM4; the topoisomerase inhibitor comprises camptothecin derivative SN-38, exatecan, and DXd; the alkylating chemotherapeutic agent and other compounds having an alkylated form comprises nitrogen mustard, ethyleneimine compounds, alkyl sulfonates, nitrosourea, cisplatin, and dacarbazine; the antimetabolitic agent comprises folic acid, purine, or pyrimidine antagonist; the mitotic inhibitor comprises vinca alkaloids and podophyllotoxin derivatives; the cytotoxic antibiotic comprises anthracycline antibiotics, actinomycin, and bleomycin; and the preferred small molecule toxin compound is eribulin.

24. The above-mentioned cleavable linker comprises a cleavable peptide portion and at least one spacer, Furthermore, the cleavable peptide portion includes an amino acid unit containing the amino acid sequence of Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG), which can be cleaved by cathepsin B. The ADC according to any one of claims 19 to 23, wherein the spacer comprises a spacer bound to the antibody and / or a second spacer bound to the small molecule toxin compound, the spacer bound to the antibody is a hydrophilic spacer containing one or more polyethylene glycols (PEGs), and the second spacer contains a p-aminobenzyl unit.

25. The ADC according to claim 24, wherein the above amino acid unit comprises VC and GGFG, the hydrophilic spacer comprises two PEG portions, the hydrophilic spacer and maleimide (Mal) form a Mal-spacer that binds to the antibody, and the second spacer comprises p-aminobenzylcarbonyl (PAB).

26. The ADC according to claim 24 or 25, wherein the cleavable linker comprises a Mal-spacer and a cleavable peptide moiety, the Mal-spacer comprising two PEGs, and the cleavable peptide moiety comprising dipeptide VC and tetrapeptide GGFG; or the ADC according to claim 24 or 25, wherein the cleavable linker comprises a Mal-spacer, a cleavable peptide moiety and a second spacer, the Mal-spacer comprising two PEGs, the cleavable peptide moiety comprising dipeptide VC, and the second spacer comprising PAB.

27. The above-mentioned cuttable linker is Mal-(PEG) ２ -VC, Mal- (PEG) ２ -GGFG, or Mal-(PEG) ２ - ADC according to any one of claims 24 to 26, comprising VC-PAB.

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

29. The above-mentioned severable linker is Mal-(PEG). ２ - Containing GGFG, the drug payload is obtained by linking the cleavable peptide portion GGFG of the cleavable linker to the C-35 amine of eribulin via a carboxyl group, resulting in the compound Mal-(PEG). ２ -Containing GGFG-eribulin; or The above-mentioned severable linker is Mal-(PEG). ２ -VC-PAB comprising the compound Mal-(PEG) obtained by linking the cleavable linker to the C-35 amine of eribulin via the second spacer PAB, wherein the drug payload is ２ The ADC according to claim 28, comprising -VC-PAB-eribulin.

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

31. An anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, or a nucleic acid encoding the antibody component of an ADC according to any one of claims 19 to 30.

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

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

34. A method for preparing an anti-HER2 antibody or its antigen-binding fragment according to any one of claims 1 to 9, an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, or an antibody component of an ADC according to any one of claims 19 to 30, using the host cells described in claim 33, comprising: (1) expressing the anti-HER2 antibody or its antigen-binding fragment, the anti-HER2 biparatopic antibody, or the antibody component of the ADC in the host cells; and (2) isolating the anti-HER2 antibody or its antigen-binding fragment, the anti-HER2 biparatopic antibody, or the antibody component of the ADC from the host cells or cell culture.

35. A method for preparing an ADC according to any one of claims 19 to 30, comprising reacting a nucleophilic or electrophilic group of the above small molecule toxin compound with a cleavable linker to form a drug payload via a covalent bond, and subsequently reacting it with a nucleophilic or electrophilic group of the above antibody, or preferably reacting a free cysteine ​​residue generated by reduction of an interchain disulfide bond in the hinge region of the above antibody with a reactive functional group of the drug payload to form a covalent bond, wherein the reactive functional group comprises a maleimide group.

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

37. A kit comprising an effective amount of an anti-HER2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, an anti-HER2 biparatopic ADC according to any one of claims 19 to 30, or a pharmaceutical composition according to claim 36, and optionally at least one additional tumor treatment agent, Furthermore, the kit includes the above additional tumor treatment agents: 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 HER2-expressing cancer, comprising applying an effective amount of an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, an anti-HER2 biparatopic ADC according to any one of claims 19 to 30, a pharmaceutical composition according to claim 36, or a kit according to claim 37 to a subject in need thereof.

39. The cancers mentioned above 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 mentioned above also include cancers of all stages, such as early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission, and the cancers mentioned above further include HER2-overexpressing cancers, cancers expressing relatively low levels of HER2, cancers that are unresponsive or poorly responsive to existing HER2-targeted therapies, and / or cancers that have acquired resistance to or relapsed to existing HER2-targeted therapies, and the existing HER2-targeted therapies mentioned above include trastuzumab, pertuzumab, T-DM1, and DS-8201. The method according to claim 38, wherein the subject may be a human, a non-human primate, or another mammal such as a dog, mouse, or rat, and further comprises a patient who is ineligible for or refractory to existing HER2-targeted therapies, or who has developed resistance to or relapsed to 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 who may respond to treatment with an ADC according to any one of claims 19 to 30, comprising incubating an anti-HER2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or an anti-HER2 biparatopic antibody according to any one of claims 10 to 18, with the sample or a biological specimen isolated from the patient, and detecting whether the antibody binds to the sample or the biological specimen.

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