WO2024251240A1 - 抗her2互补性双特异抗体-药物偶联物及其制备方法和用途 - Google Patents

抗her2互补性双特异抗体-药物偶联物及其制备方法和用途 Download PDF

Info

Publication number
WO2024251240A1
WO2024251240A1 PCT/CN2024/097980 CN2024097980W WO2024251240A1 WO 2024251240 A1 WO2024251240 A1 WO 2024251240A1 CN 2024097980 W CN2024097980 W CN 2024097980W WO 2024251240 A1 WO2024251240 A1 WO 2024251240A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
her2
amino acid
antibody
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2024/097980
Other languages
English (en)
French (fr)
Inventor
李约翰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Latticon Suzhou Biopharmaceuticals Co Ltd
Original Assignee
Latticon Suzhou Biopharmaceuticals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Latticon Suzhou Biopharmaceuticals Co Ltd filed Critical Latticon Suzhou Biopharmaceuticals Co Ltd
Priority to CN202480038349.6A priority Critical patent/CN121311508A/zh
Priority to EP24818770.0A priority patent/EP4725969A1/en
Publication of WO2024251240A1 publication Critical patent/WO2024251240A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-HER2 antibody or an antigen-binding fragment thereof, and an anti-HER2 complementary bispecific antibody constructed therefrom and an antibody-drug conjugate comprising the same.
  • the present invention also provides a method for preparing the complementary bispecific antibody and its antibody-drug conjugate, and a method for treating a disease associated with abnormal HER2 expression.
  • HER2 Human Epidermal Growth Factor Receptor 2
  • ErbB/HER family receptors activate downstream signal transduction pathways mediated by dimerization, and thus participate in the regulation of cell growth, differentiation and survival.
  • HER2 is a transmembrane glycoprotein with a molecular weight of about 185 kDa, which consists of an extracellular domain, a transmembrane domain and an intracellular tyrosine kinase domain.
  • ErbB2/HER2 gene mutations and/or gene amplification can lead to HER2 overexpression, which is manifested as IHC 3 + or IHC 2 + /FISH + in IHC and FISH detection.
  • Clinical studies have shown that HER2 is overexpressed in a variety of cancers (such as breast cancer, ovarian cancer, endometrial cancer, gastric cancer, prostate cancer, lung cancer, etc.), and this overexpression is associated with tumor aggressiveness and poor prognosis.
  • HER2-targeted therapeutic drugs have been approved for the treatment of HER2-overexpressing breast cancer and gastric cancer. These drugs have significantly prolonged the survival of patients; however, a considerable number of cancer patients still do not respond to these therapies.
  • existing HER2-targeted therapies have the following defects that limit their clinical efficacy and lead to safety risks:
  • HER2 targeted therapies lack effective killing power against tumor cells with relatively low HER2 expression, where relatively low HER2 expression refers to 2 + / FISH- or IHC 1+ in IHC detection (defined as "HER2 low expression”); since HER2 expression is highly heterogeneous in HER2-overexpressing tumors, tumor cells with low HER2 expression in the tumors are easily resistant to existing therapies, resulting in disease progression in most cancer patients due to drug resistance after 6 to 12 months of receiving existing HER2 targeted therapies;
  • HER2-mediated signal transduction pathways are crucial for maintaining the survival of cardiomyocytes, repairing cardiomyocyte damage, and maintaining the integrity of cardiomyocyte function.
  • approved anti-HER2 antibodies, ADCs, or small molecule inhibitors can block or disrupt the HER2 signal transduction pathway, and therefore have toxic side effects that can induce cardiac dysfunction (cardiotoxicity).
  • trastuzumab and pertuzumab may both cause a decrease in left ventricular ejection fraction (LVEF) and congestive heart failure during treatment.
  • LVEF left ventricular ejection fraction
  • trastuzumab has a higher risk of inducing heart failure when administered simultaneously with anthracyclines. This leads to the need to strictly screen the population with indications or to perform preventive cardiac protection treatment on patients before using these drugs for treatment.
  • the present invention provides a complementary bispecific antibody-drug conjugate targeting HER2 (HER2-targeting Biparatopic ADC).
  • the complementary bispecific ADC can specifically bind to two different epitopes in the extracellular domain of HER2 (also called "extracellular region"), so it can effectively crosslink the cell surface HER2 to form a matrix-like polymer, thereby inducing rapid and efficient endocytosis, and guiding the endosome after endocytosis to change from the usual recycling transport pathway to the lysosomal transport pathway, thereby making the ADC bound to the cell surface be efficiently transported (almost 100%) into the lysosome for degradation, thereby releasing more small molecule toxins into the cytoplasm to exert cytotoxicity and subsequent bystander killing effects.
  • the complementary bispecific ADC has a broader spectrum of targeted killing activity, which can not only kill cancer cells that overexpress HER2, but also directly kill cancer cells that underexpress HER2 (IHC 2 + / FISH- or IHC 1+ ), thereby greatly reducing the probability of drug resistance due to the heterogeneity of HER2 expression; on the other hand, the antigen binding epitope of the complementary bispecific ADC is different from that of Trastuzumab or Pertuzumab, and it will not interfere with the dimerization of HER2 or affect the regulation of the downstream signal transduction pathway of HER2, and therefore will not affect the normal biological function of HER2 in cardiomyocytes.
  • the present invention provides an isolated anti-HER2 antibody or antigen-binding fragment thereof, wherein the anti-HER2 antibody or antigen-binding fragment thereof can specifically bind to the extracellular domain of HER2, has a high binding activity or binding affinity (e.g., a binding affinity constant [ KD ] value of ⁇ 1 ⁇ 10-8 M, ⁇ 5 ⁇ 10-9 M, or ⁇ 1 ⁇ 10-9 M) to tumor cells expressing HER2, and does not cross-bind with other members of the ErbB/HER family (including EGFR, HER3, and HER4).
  • the anti-HER2 antibody or antigen-binding fragment thereof does not have an inhibitory or activating effect on HER2 and its downstream signal transduction pathways.
  • the anti-HER2 antibody includes a murine anti-HER2 antibody or a chimeric antibody and/or a humanized antibody derived therefrom and an optimized antibody thereof.
  • the anti-HER2 antibody or its antigen-binding fragment specifically binds to subdomains 1, 3 and/or 4 of the HER2 extracellular region, preferably HER2 extracellular region subdomain 1 or 3.
  • the two anti-HER2 antibodies or their antigen-binding fragments used to construct an anti-HER2 complementary bispecific antibody specifically recognize different epitopes in the HER2 extracellular region, and there is no competitive binding relationship between them.
  • the present invention provides an anti-HER2 complementary bispecific antibody, which comprises a first antigen binding domain and a second antigen binding domain, wherein the first and second antigen binding domains specifically bind to different epitopes of HER2, so that the bispecific antibody can cross-link HER2 on the cell surface to form clusters, and the formation of clusters is subject to the expression abundance of HER2 on the cell surface; the formation of clusters will trigger its rapid endocytosis by cells and transport to lysosomes for degradation.
  • the complementary bispecific antibody has a significantly improved HER2 internalization rate and a higher lysosomal transport/degradation efficiency.
  • the first and second antigen binding domains of the anti-HER2 complementary bispecific antibody specifically bind to different (non-overlapping or non-competing) epitopes of HER2, and the epitopes include sequences located in HER2 extracellular subdomains 1, 3, and/or 4.
  • the first antigen binding domain specifically binds to HER2 extracellular subdomain 3
  • the second antigen binding domain specifically binds to HER2 extracellular subdomain 1, and the bound epitopes are different from the antigen binding epitopes of Trastuzumab or Pertuzumab.
  • the complementary bispecific antibody has no inhibitory or activating effect on cell surface HER2 and its downstream signal transduction pathways.
  • the complementary bispecific antibody triggers its rapid internalization by cells and efficient lysosomal transport after binding to and cross-linking HER2 on the cell surface.
  • the complementary bispecific antibody can induce degradation of cell surface HER2.
  • the complementary bispecific antibody cross-links cell surface HER2, causing it to be internalized and transported to lysosomes for degradation, resulting in a significant decrease in the abundance of cell surface HER2, thereby effectively inhibiting the proliferation of HER2-overexpressing tumor cells.
  • the present invention provides an ADC, which couples a small molecule toxin compound to the anti-HER2 complementary bispecific antibody of the present invention through a linker.
  • the ADC can specifically bind/cross-link HER2 expressed on the surface of tumor cells and aggregate into "clusters", so that the ADC is quickly internalized and transported to the lysosome for degradation, which significantly improves the efficiency of the small molecule toxin compound in the ADC entering the tumor cells and being released in the tumor cells. Therefore, the complementary bispecific ADC has a broader spectrum of tumor cell killing activity than T-DM1 and/or DS-8201, that is, the ADC has direct killing activity against both HER2 overexpressing and HER2 underexpressing tumor cells.
  • the ADC can be represented by formula (I): Ab-(LD)p (I)
  • Ab represents the anti-HER2 complementary bispecific antibody of the present invention
  • D represents a small molecule toxin compound (Drug);
  • L represents a cleavable linker that couples Ab to D
  • p represents the number of copies of (L-D) coupled to Ab, which ranges from 2 to 8.
  • the small molecule toxin compound includes cytotoxins and chemotherapeutic drugs.
  • the small molecule toxin compound is a cytotoxin, including tubulin inhibitors and DNA damaging agents; preferably, the tubulin inhibitors include Eribulin, Auristatins derivatives (e.g., MMAE, MMAF, MMAD), Tubulysins, Cryptomycins and Maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4), and the DNA damaging agents include topoisomerase inhibitors (e.g., camptothecin derivatives SN-38, Exatecan and DXd [Exatecan derivative for ADC]), pyrrolobenzodiazepines (PBD), Calichaemicin and its derivatives (e.g., N-acetyl Calichaemicin [CMC]), and Duocarmycin.
  • the small molecule toxin compound is Eribulin.
  • the cleavable linker can be any linker comprising a cleavable portion, and the cleavable portion comprises any cleavable chemical bond.
  • the cleavable linker comprises a cleavable peptide portion, which can be cleaved by an intracellular peptidase or protease, and the cleavable peptide portion comprises an amino acid unit, and the amino acid unit comprises a dipeptide, a tripeptide, or a tetrapeptide.
  • the cleavable linker may include at least one spacer (Spacer) for conjugating the anti-HER2 complementary bispecific antibody (Ab) of the present invention to a small molecule toxin compound (D), and the spacer comprises a spacer conjugated to the antibody, and/or a second spacer conjugated to the small molecule toxin compound.
  • the spacer conjugated to the antibody is hydrophilic, and an exemplary spacer comprises polyethylene glycol (PEG).
  • the spacer is connected to the anti-HER2 complementary bispecific antibody of the present invention via butylene diimide (Mal).
  • the second spacer conjugated to the small molecule toxin compound is used to connect the cleavable portion (e.g., cleavable peptide) of the cleavable linker to the small molecule toxin compound.
  • the second spacer conjugated to the small molecule toxin compound has the property of self-immolation, and the self-immolation spacer comprises a para-aminobenzyl unit.
  • the cleavable linker comprises a Mal-spacer and a cleavable peptide portion.
  • the Mal-spacer of the cleavable linker is coupled to one or more amino acid residues of the antibody portion in the ADC of the present invention.
  • the maleimide group of the Mal-spacer can react with the sulfhydryl group of the cysteine residue at a specific position in the constant region and/or variable region of the antibody.
  • the second spacer of the cleavable linker is coupled to the small molecule toxin compound portion in the ADC of the present invention, and the small molecule toxin compound is eribulin or its derivatives.
  • the cleavable portion of the cleavable linker (e.g., a cleavable peptide) can be directly conjugated to the small molecule toxin compound portion of the ADC, and the small molecule toxin compound is eribulin or Its derivatives.
  • p is 2 to 8, for example 4 to 8.
  • p may be an integer greater than 0 or a non-integer.
  • the present invention relates to an isolated nucleic acid molecule (also referred to as a "polynucleotide”) encoding an anti-HER2 complementary bispecific antibody and its corresponding monospecific antibody or antigen-binding fragment thereof, as well as an expression vector comprising the nucleic acid and a host cell comprising the nucleic acid or the expression vector.
  • the present invention also relates to a method for preparing the monospecific anti-HER2 antibody or its antigen-binding fragment described herein and the anti-HER2 complementary bispecific antibody using the host cell, the method comprising culturing the host cell and recovering the antibody or its antigen-binding fragment from the culture medium.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the monospecific anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody, or the anti-HER2 complementary bispecific antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
  • the present invention relates to a drug kit comprising an effective amount of the anti-HER2 monospecific antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, the anti-HER2 complementary bispecific antibody-drug conjugate, or the pharmaceutical composition of the present invention, and optionally at least one additional tumor therapeutic agent.
  • the present invention relates to a method for treating a HER2-expressing tumor in a subject, the method comprising administering to a subject in need thereof an anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition or a kit of the present invention.
  • the present invention relates to the use of the anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition or a kit of medicine described herein for preparing a drug for treating a HER2-expressing tumor in a subject.
  • the present invention relates to the anti-HER2 complementary bispecific antibody, an anti-HER2 complementary bispecific antibody-drug conjugate, a pharmaceutical composition or a kit of medicine described herein for treating a HER2-expressing tumor in a subject.
  • the tumor includes a tumor with HER2 overexpression (IHC 3 + , or IHC 2 + /FISH + ) and/or a tumor with HER2 underexpression (IHC 2 + /FISH - , or IHC 1 + ).
  • the anti-HER2 complementary bispecific antibody-drug conjugate or its pharmaceutical composition has a killing effect on tumor cells that overexpress HER2 and/or underexpress HER2.
  • the subject is a mammal.
  • the tumor includes 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 tumor can be HER2 overexpressed or underexpressed.
  • the tumor is a tumor that is resistant to existing HER2 targeted therapeutic agents. In some embodiments, the tumor is a tumor that is resistant to HER2 targeted therapeutic agents including Trastuzumab, Pertuzumab, T-DM1 and/or DS-8201. In one embodiment, the tumor is a tumor that does not respond or responds poorly to targeted therapeutic agents including Trastuzumab, Pertuzumab, T-DM1, DS-8201 and/or taxanes (e.g., Paclitaxel, Docetaxel, Cabazitaxel, etc.).
  • taxanes e.g., Paclitaxel, Docetaxel, Cabazitaxel, etc.
  • the subject includes patients who are not suitable for or refractory to existing HER2 targeted therapies, or who develop drug resistance or relapse after receiving existing HER2 targeted therapies.
  • the existing HER2 targeted therapies include treatment with Trastuzumab, Pertuzumab, T-DM1, and/or DS-8201.
  • 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 respond to treatment with the anti-HER2 complementary bispecific ADC described in the present invention, comprising incubating the anti-HER2 monospecific antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody described in the present invention with the sample or a biological sample isolated from the patient, and detecting whether the antibody binds to the sample or the biological sample.
  • FIG. 1 ELISA was used to detect the binding activity of each anti-HER2 chimeric antibody to human ErbB/HER family proteins EGFR, HER2, HER3 and HER4, where the control antibodies included Trastuzumab, Cetuximab and Patritumab.
  • FIG. 1 Flow cytometry was used to detect the binding activity of anti-HER2 chimeric antibodies to NCI-N87 cells overexpressing HER2, where the control antibody was Trastuzumab.
  • Figure 3 Schematic diagram of the structure of the human-mouse chimeric HER2 extracellular region recombinant protein (Figure 3A), and the ELISA method to determine the HER2 extracellular region subdomain where the antigen binding epitope of the anti-HER2 chimeric antibody is located ( Figure 3B).
  • Figure 4 The competitive ELISA method was used to detect whether there was a competitive binding relationship between the anti-HER2 chimeric antibodies or between them and the control antibody Trastuzumab, wherein Figure 4A shows the results of biotin-labeled mAb2164 competing with mAb2117, mAb2126, mAb2170 and Trastuzumab for binding to HER2 recombinant protein, respectively, and Figure 4B shows the results of mAb2117 competing with biotin-labeled mAb2128 or biotin-labeled mAb2126 for binding to HER2 recombinant protein, and mAb2164 competing with biotin-labeled mAb2128 for binding to HER2 recombinant protein.
  • Figure 5 Effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on phosphorylation of HER2 intracellular domain Y1248 site in SKBR-3 cells, wherein control antibodies include Trastuzumab and Pertuzumab.
  • FIG. 6 Effects of anti-HER2 chimeric antibodies mAb2117 and mAb2126 on NRG-1-induced AKT phosphorylation in T47D cells, wherein control antibodies include Trastuzumab and Pertuzumab.
  • Figure 7 Flow cytometry was used to detect the binding activity of optimized anti-HER2 single-chain antibody molecules containing single-point/multi-point mutations to BT474 cells ( Figure 7A) and RT-112 cells ( Figure 7B), respectively, where the control antibody was the parent single-chain antibody Hu2117HK.
  • FIG. 8 Flow cytometry was used to detect the binding activity of optimized anti-HER2 single-chain antibody molecules containing combined mutations (Hu2117-HK203, Hu2117-HK303, Hu2117-HK304, Hu2117-HK309, Hu2117-HK310) to BT474 cells and RT-112 cells, respectively, where the control antibody was the parent single-chain antibody Hu2117HK.
  • FIG. 9 Schematic diagram of the structure of an exemplary anti-HER2 complementary bispecific antibody 04BS-109-WT.
  • the configuration of the exemplary complementary bispecific antibody is a DVD-IgG structure, wherein the Fv domain is the variable region sequence of Hu2117-HK304, the IgG domain is the full-length sequence of Hu2126-H2K1-L71-H72b-Mu14, the Fv domain heavy chain variable region is connected to the heavy chain of the IgG domain through a linker (G 4 S) 1 , the Fv domain light chain variable region is connected to the light chain of the IgG domain through a linker (G 4 S) 3 , and the antibody heavy chain Fc region contains L234F/L235E/P331S (EU Numbering) mutations.
  • the Fv domain is the variable region sequence of Hu2117-HK304
  • the IgG domain is the full-length sequence of Hu2126-H2K1-L71-H72b-Mu14
  • FIG. 10 Flow cytometry was used to determine the co-binding or competitive binding relationship between the anti-HER2 humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) and BT474 cells.
  • FIG. 11 Flow cytometry was used to detect the endocytosis reaction of the anti-HER2 complementary bispecific antibody 04BS-109-WT and its mutant molecules in cell lines (BT474, JIMT-1, RT-112) expressing different levels of HER2, wherein the control antibodies included Trastuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibody 04BS-1123-ST06.
  • FIG. 12 Confocal microscopy was used to detect the lysosomal transport efficiency of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 after being internalized by SKBR-3 cells, wherein the control antibodies included Trastuzumab and human IgG isotype antibodies; the arrows in the figure indicate that the fluorescent signal sites of the antibody and the fluorescent signal sites of the lysosome overlap with each other, indicating that the cross-linked aggregates or clusters formed by the complementary bispecific antibody and HER2 were internalized by the cells and transported into the lysosomes; T represents the antibody incubation time (hours).
  • FIG. 13 Western-blot method was used to detect the degradation reaction of HER2 protein in BT474 cells caused by the anti-HER2 complementary bispecific antibody 04BS-1123-ST06, wherein the control antibodies included Trastuzumab and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibodies.
  • the control antibodies included Trastuzumab and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibodies.
  • Figure 14 Effect of anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on the in vitro proliferation of BT474 cells, wherein control antibodies include Trastuzumab, Pertuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibodies.
  • control antibodies include Trastuzumab, Pertuzumab, and the monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 (abbreviated as Hu2126-7172b-Mu14) corresponding to the complementary bispecific antibodies.
  • Figure 15 The reporter gene method was used to detect the effects of anti-HER2 monospecific antibodies (Figure 15A) and anti-HER2 complementary bispecific antibodies (Figure 15B) on NRG-1-induced HER2/HER4 dimerization, wherein the control antibodies included Trastuzumab and Pertuzumab.
  • FIG. 16 Western-blot method was used to detect the effect of anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes, wherein the control antibodies included Trastuzumab and Pertuzumab.
  • FIG. 1 The ADCC activity of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 was detected by the reporter gene method, wherein the positive control antibody was Trastuzumab.
  • FIG. 18 The ELISA method was used to detect the binding specificity of the anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) to human ErbB/HER family members (including EGFR, HER2, HER3 and HER4).
  • ST06-VCP-Eribulin anti-HER2 complementary bispecific ADC
  • FIG. 19 In vitro cell killing assay was used to detect the proliferation inhibitory activity of anti-HER2 complementary bispecific ADCs (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) and the benchmark molecule DS-8201 in tumor cell lines expressing different levels of HER2.
  • anti-HER2 complementary bispecific ADCs including ST06-GGFG-Eribulin and ST06-VCP-Eribulin
  • benchmark DS-8201 in tumor cell lines expressing different levels of HER2.
  • Figure 20 The bystander effect of the anti-HER2 complementary bispecific ADC was detected by an in vitro cell killing experiment, wherein Figure 20A shows the result of detecting the killing activity of BT474 cells by ST06-GGFG-Eribulin after 3 days of treatment of BT474 cells, and the culture supernatant (BT474-conditioned Medium) was collected for incubation of MDA-MB-468 cells, and the survival of MDA-MB-468 cells was detected after 3 days to detect the bystander killing effect of the ADC, wherein the control group was the survival of MDA-MB-468 cells incubated with freshly prepared ST06-GGFG-Eribulin performed simultaneously.
  • Figure 20A shows the result of detecting the killing activity of BT474 cells by ST06-GGFG-Eribulin after 3 days of treatment of BT474 cells, and the culture supernatant (BT474-conditioned Medium) was collected for incubation of MDA-MB-468 cells, and the survival of MDA-MB-468 cells was detected after 3
  • FIG20B shows the cell killing activity of ST06-GGFG-Eribulin detected by flow cytometry in BT474 cells overexpressing HER2 and Jurkat cells not expressing HER2 when cultured alone or in co-culture.
  • the numbers shown in the upper left and lower right corners of the figure are the viable cell counts of BT474 cells and Jurkat cells, respectively.
  • Figure 21 The in vivo anti-tumor activity of the anti-HER2 complementary bispecific ADC (ST06-GGFG-Eribulin) was detected in mouse subcutaneous xenograft tumor models constructed based on tumor cell lines NCI-N87 ( Figure 21A), JIMT-1 ( Figure 21B) and RT-112 ( Figure 21C), where the control group included a mixture of antibodies and small molecule compounds (ADMix) group, DS-8201 group and vehicle group (Vehicle), and all tumor-bearing mice were dosed by tail vein injection.
  • the arrows in the figure indicate the time points of administration.
  • Figure 22 The in vivo anti-tumor activity of the anti-HER2 complementary bispecific ADC (ST06-GGFG-Eribulin) was detected using a mouse subcutaneous xenograft tumor model with acquired resistance to DS-8201, where the control groups included the DS-8201 group and the vehicle group (Vehicle).
  • HER2 and "HER2 receptor” are used interchangeably, and the protein is also referred to as ErbB2, c-ERB2, c-ERB-2, NEU, HER-2/neu, p185 (erbB2) or CD340. Unless otherwise specified that it is from a non-human species, for example, “mouse HER2”, “monkey HER2”, etc., "HER2” as used herein refers to any natural form of human HER2, which may have an amino acid sequence as shown in SEQ ID NO: 234 and/or a full-length HER2 amino acid sequence as shown in NCBI Accession No.
  • NP_004439.2 may also be naturally expressed by cells (including tumor cells) or expressed by cells transfected with a HER2 gene or cDNA.
  • the term includes naturally occurring HER2 allelic variants and splice variants, isoforms, homologs and species homologs.
  • HER2 can be isolated from the human body or can be produced by recombinant or synthetic methods.
  • the extracellular domain of HER2 is composed of four subdomains, namely subdomain 1 (D1, approximately amino acid residues 1-195), subdomain 2 (D2, approximately amino acid residues 196-319), subdomain 3 (D3, approximately amino acid residues 320-488), and subdomain 4 (D4, approximately amino acid residues 489-630) (residue numbering without signal peptide); among them, D2 and D4 are cysteine-rich domains responsible for receptor dimerization (Garrett et al., Mol Cell 2003, 11:495-505; Cho et al., Nature 2003, 421:756-760; Franklin et al., Cancer Cell 2004, 5:317-328).
  • cells expressing HER2 may be naturally occurring cells or cell lines (eg, tumor cells), or may be recombinantly produced by introducing a nucleic acid encoding HER2 into a host cell.
  • bispecific antibody or “bispecific antibody” is intended to include any antibody or antigen-binding fragment that can specifically bind to two different antigen epitopes, comprising two independent antigen-binding domains, each with a unique antigen-binding specificity.
  • the "complementary bispecific antibody” described herein specifically refers to a class of bispecific antibodies, whose first antigen-binding domain and second antigen-binding domain respectively bind to different epitopes on the same antigen.
  • a monospecific antibody refers to an antibody or antigen-binding fragment with only one binding specificity, i.e., the antigen-binding domain of a monospecific antibody binds to a single epitope of a single antigen.
  • examples of the monospecific antibody include the anti-HER2 monospecific antibodies of the present invention.
  • antigen binding domain or “antigen binding region” or “epitope binding domain” or “antigen binding polypeptide” are used interchangeably, and refer to a specific region on an antibody or antigen binding fragment or derivative thereof, which is directly involved in the specific interaction with the target antigen, such as interacting with the target antigen through binding, steric hindrance, stabilization/destabilization, spatial distribution, etc. to achieve dynamic equilibrium.
  • antigen binding domain also refers to a specific region on the antibody or antigen binding fragment or derivative thereof, which interacts with a specific epitope on HER2, and achieves dynamic equilibrium between the binding between the two through binding, steric hindrance, stabilization/destabilization, spatial distribution, etc.
  • Antibody refers to a polypeptide or protein that is generally encoded by an immunoglobulin gene or multiple immunoglobulin genes, or a fragment thereof, and is capable of specifically recognizing and binding to an antigen.
  • the recognized immunoglobulin genes include ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as numerous immunoglobulin variable region genes.
  • Light chains are classified as ⁇ or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin class (Class) or isotype (Isotype) IgG, IgM, IgA, IgD, and IgE, respectively, several of which can be further divided into subclasses (Subclass), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the typical immunoglobulin (e.g., antibody) structural unit is a tetramer, each of which is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kD) and a "heavy" chain (about 50-70 kD).
  • the N-terminal domain of each chain defines a variable (V) region of approximately 100 to 110 or more amino acids that is primarily responsible for antigen recognition.
  • the antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region typically includes three domains, CH1, CH2, and CH3.
  • the light chain consists of a light chain variable region (VL) and a light chain constant domain (CL), wherein the light chain constant domain typically includes one domain, CL.
  • the pairing of VH and VL together forms a single antigen binding site. Endogenous VL is encoded by gene segments V (variable) and J (joining), and endogenous VH is encoded by V, D (diversity), and J.
  • VL and VH include a hypervariable region (Region of Hypervariability) or complementarity determining region (CDR) and a framework region (FR).
  • the terms "variable region” or “V region” are used interchangeably and refer to a heavy chain variable region or a light chain variable region arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus.
  • J region refers to a subsequence of a variable region encoding the C-terminal portion comprising CDR3 and FR4.
  • the V region or J region may be naturally occurring, recombinant or synthetic.
  • the antibody light chain variable region and/or the antibody heavy chain variable region may sometimes be collectively referred to as an "antibody variable region", and the antibody light chain and/or the antibody heavy chain may be collectively referred to as an "antibody chain”.
  • the FRs of the antibodies or antigen-binding fragments thereof provided herein may be identical to human germline sequences, or may be natural or artificially modified.
  • CDRs and FRs can be determined using a variety of definitions known in the art, for example, Kabat, Chothia, IMGT and Contact (see: Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition 1991, NIH Publication No.
  • the present invention includes any definition method to determine the CDRs in the anti-HER2 complementary bispecific antibody of the present invention or the anti-HER2 antibody of the present invention or its antigen-binding fragment, and Table 1 shows the position numbering of the antibody CDR amino acid sequence determined using different definition methods. The exact number of amino acid residues covering a particular CDR varies with the sequence of the CDR. In the case of clarifying the amino acid sequence of the antibody variable region, a person skilled in the art can determine the CDR of the antibody by conventional methods including but not limited to the definition.
  • CDR 1 determined by different definition methods 1 The numbering of all CDR definitions in Table 1 is based on the numbering system proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 1991, fifth edition, NIH publication number 91-3242).
  • Kabat et al. also defined a numbering system for variable region sequences that can be applied to any antibody.
  • One of ordinary skill in the art can clearly apply this "Kabat numbering" system to the variable region sequence of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence.
  • the numbering of specific amino acid residue positions in the variable region of the antigen-binding domain of the anti-HER2 antibody, anti-HER2 complementary bispecific antibody, or complementary bispecific ADC described in the present invention is determined according to the Kabat numbering system.
  • Antibodies exist in the form of complete immunoglobulins, or in the form of many fragments produced by digestion with a variety of peptidases. Although various antibody fragments are defined according to the digestion of complete antibodies, it will be appreciated by the technician that such fragments can be synthesized by chemical cleavage methods or by using recombinant DNA methods.
  • the "antigen binding fragment” (or simply referred to as “antibody part” or “antibody fragment”) of the term antibody refers to an antibody part containing one or more CDRs or any other antibody fragment that can be combined with an antigen (e.g., the extracellular domain of HER2 or HER2) but does not have a complete antibody structure.
  • Antigen binding fragments can have the same activity of specifically binding antigens as complete antibodies. Preferred antigen binding fragments also retain the ability to internalize into cells expressing target antigens. In certain embodiments, an antigen binding fragment can contain one or more CDRs from a particular human antibody, grafted to the framework region from one or more different human antibodies.
  • Antigen-binding fragments include, but are not limited to: (i) a "Fab” fragment, which is a monovalent antibody fragment consisting of the VH, VL, CL and CH1 domains; (ii) a "F(ab')2" fragment, which is a bivalent fragment consisting of two Fab fragments connected by a disulfide bond in the hinge region; (iii) a "Fv” fragment, which is composed of the VL and VH domains of a single antibody arm and is the smallest antibody fragment containing a complete antigen-binding site; (iv) a "Fd” fragment, which is composed of the VH and CH1 domains; (v) a “single-chain Fv antibody (scFv)” or “single-chain antibody”, which refers to an engineered antibody in which the light chain variable region is directly connected to the heavy chain variable region or connected by a peptide chain (Huston et al., Proc Natl Acad Sci USA 1988,
  • Single-domain antibodies are independent immunoglobulin domains;
  • “Diabodies” are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow the two domains on the same chain to pair, thus forcing the two domains to pair with the complementary domains of another chain to form two antigen-binding sites (Holliger et al., Proc Natl Acad Sci USA 1993, 90:6444-6448; Poljak et al., Structure 1994, 2:1121-1123; EP404097; WO93/11161).
  • the term "Fc region” or "Fc domain” refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region, such as an immunoglobulin heavy chain constant region other than the first constant region (CH1).
  • the Fc region may include immunoglobulin domains CH2 and CH3 and a hinge region between CH1 and CH2.
  • the Fc region used herein includes a native sequence Fc region and/or an Fc region variant, and may be part of an anti-HER2 antibody, an anti-HER2 complementary bispecific antibody, or an ADC thereof of the present invention.
  • the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is generally defined as containing a cysteine residue at position 226 or a proline residue at position 230 at its amino terminus, according to the EU numbering system/scheme, as found in Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242.
  • anti-HER2 antibody or “antibody that specifically binds to HER2” refers to any form of antibody or fragment thereof that specifically binds to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as the fragment specifically binds to HER2. Just bind to HER2.
  • the term "specific binding” or “binding specificity” or “specific for” or “binding” refers to a binding reaction that determines the presence of a target molecule (e.g., an antigen) in a heterogeneous population of proteins and other biological products (e.g., biological samples such as blood, serum, plasma or tissue samples), that is, the binding is selective for the target molecule and can distinguish those undesirable or non-specific interactions.
  • a target molecule e.g., an antigen
  • biological samples e.g., biological samples such as blood, serum, plasma or tissue samples
  • this selection can be achieved by removing antibodies that cross-bind to other ErbB/HER family members.
  • a variety of immunoassays can be used to select antibodies that specifically react with a particular protein, such as an ELISA assay.
  • the specific or selective binding reaction of an antibody or binding agent to an antigen will produce a signal at least twice the background, more typically at least 10-100 times the background, and will not substantially bind to other antigens present in the sample in significant amounts.
  • the equilibrium dissociation constant ( KD ) for specific binding of an antibody to a target antigen is ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for natural mutations that may be present in small amounts. Monoclonal antibodies exhibit a high degree of binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al., Nature 1975, 256:495, or by recombinant DNA methods (see US4816567), or can be isolated from phage antibody libraries, for example, by reference to the techniques described in Clackson et al., Nature 1991, 352:624-628; Marks et al., J Mol Biol 1991, 222:581-597.
  • chimeric antibody refers to an antibody containing sequences derived from two different antibodies (e.g., US4816567), which are typically derived from different species.
  • a chimeric antibody comprises human and rodent antibody fragments, typically a human constant region and a mouse variable region.
  • Methods for producing chimeric antibodies include recombinant DNA and gene transfection techniques known to those of ordinary skill in the art (e.g., Morrison et al., Proc Natl Acad Sci USA 1984, 81: 6851-6855; US 5202238 and US 5204244).
  • humanized antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, a FR region derived from a human, and a constant region derived from a human.
  • the humanized antibody optionally also comprises at least a portion of the constant region of a human immunoglobulin. Since the humanized antibody or antigen-binding fragment has reduced immunogenicity, it can be used as a therapeutic agent for administration to the human body.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig or hamster.
  • the humanized antibody or antigen-binding fragment is substantially composed of human sequences except that the CDR sequence is non-human.
  • humanized antibodies can be further modified, improved and optimized by replacing the corresponding residues in the human immunoglobulin FR framework region with residues in non-human species antibodies.
  • the FR region derived from humans may include the same amino acid sequence as the human antibody from which it comes, or it may include some amino acid changes, for example, no more than 5, 4, 3, 2, or 1 amino acid changes.
  • the amino acid changes may occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.
  • corresponding human germline sequence refers to an antibody variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a referenced human germline immunoglobulin variable region amino acid sequence when compared to all other known human germline immunoglobulin variable region amino acid sequences.
  • the corresponding human germline sequence can be a framework region alone, a complementarity determining region alone, a framework region and a complementarity determining region, a variable region, or other combinations comprising variable region sequences or subsequences. Sequence identity can be determined using methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or other alignment algorithms known in the art.
  • the corresponding human germline amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a referenced human germline immunoglobulin variable region amino acid sequence.
  • the antibody portion of the anti-HER2 antibody, anti-HER2 complementary bispecific antibody or ADC of the present invention can be selected from any one or more of the following forms, including chimeric forms, non-human forms, humanized forms or fully human forms, as long as the form can specifically bind to HER2.
  • Competitive binding and epitope grouping can also be used to determine whether different antibodies bind to the same epitope or overlapping epitopes, for example, using the method described in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory.
  • the activity of inhibiting each other's binding to the target antigen (e.g., HER2) between an antibody or its antigen-binding fragment (e.g., an antibody or antigen-binding fragment comprising a CDR and/or variable region selected from Tables 2 and 3) and other antibodies or their antigen-binding fragments is detected by a competitive binding ELISA method, and when the binding activity is reduced by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, or any percentage between the listed values), the two are determined to be competitively bound, i.e., bound to the same epitope or overlapping epitopes.
  • competitive binding can be attributed to shared or similar (e.g., partially overlapping) epitopes, or to steric hindrance caused by the binding of antibodies or antigen-binding fragments to nearby epitopes (see Morris, ed., Methods in Molecular Biology 1998, Vol. 66, pp. 55-66).
  • competitive binding can be used to group antibodies or antigen-binding fragments that bind to a shared similar epitope, for example, antibodies or antigen-binding fragments that compete for binding can be "grouped" into a group of antibodies or antigen-binding fragments with overlapping or proximal epitopes, while non-competing antibodies or antigen-binding fragments are classified as having no overlapping or proximal epitopes. Another set of antibodies or antigen-binding fragments that approach the epitope.
  • the affinity of a molecule X for its partner Y can usually be expressed in terms of an equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant ( kdis or koff ) to the association rate constant ( ka or kon ).
  • KD equilibrium dissociation constant
  • Affinity can be measured by common methods known in the art, including the methods used in the present invention.
  • antibody variant refers to an antibody polypeptide sequence containing at least one amino acid mutation in the original antibody variable region.
  • Variant can be substantially homologous or substantially identical to unmodified antibodies.
  • one or more amino acid mutations are made in the CDRs and FRs of the anti-HER2 antibody, anti-HER2 complementary bispecific antibody or ADC antibody portion of the present invention to increase the degree of humanization of the antibody or antigen-binding fragment, enhance the binding affinity or binding activity to HER2, increase the internalization rate, increase the expression amount, and/or increase stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and/or asparagine deacylation).
  • the amino acid mutations include amino acid substitutions, removals, insertions or any combination thereof.
  • non-polar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W) and methionine (Met, M);
  • 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);
  • positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K) and histidine (His, H);
  • negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E).
  • the terms “identical” or “identity” or “percent identity” or “percent sequence identity” are used interchangeably for two or more polypeptide sequences and refer to the percentage ratio of the number of amino acid residues in a candidate sequence that are identical to a reference sequence to the total number of amino acid residues in the candidate sequence when an amino acid sequence alignment is performed and after introducing the necessary intervals to maximize the number of identical amino acids. Conservative substitutions of the amino acid residues may or may not be considered identical residues.
  • Sequences may be aligned to determine the percent sequence identity of amino acid sequences using tools disclosed in the art, such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266: 383-402; Larkin et al., Bioinformatics 2007, 23: 2947-2948) and ALIGN or Megalign (DNASTAR) software.
  • tools disclosed in the art such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266: 383-402; Larkin et al., Bioinformatics 2007, 23: 2947-2948) and ALIGN or Megalign (DNASTAR) software.
  • BLASTp Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al.
  • the term "isolated” means that the protein is substantially free of other cellular components associated with it in the natural state, preferably in a homogeneous state, for example, the isolated protein can be removed from the natural or natural environment.
  • the isolated protein can be lyophilized or an aqueous solution. Usually, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography).
  • the protein is substantially purified in the isolated preparation.
  • purified means that the protein basically produces only one band in a non-reducing electrophoresis gel.
  • the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • the recombinant protein expressed in the host cell is considered to be isolated, and the same is also applicable to natural proteins separated, graded, or partially or substantially purified by any technology well known to those skilled in the art. or recombinant protein.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities (for example, an antibody that specifically binds to HER2 is substantially free of antibodies that specifically bind to antigens other than HER2).
  • the antibody that specifically binds to HER2 can have cross-binding reactivity with other antigens (such as HER2 proteins from other species, such as monkey HER2).
  • the isolated antibody can be substantially free of other cell materials and/or chemicals.
  • the recombinant polynucleotides encoding polypeptides or proteins of the present invention (such as anti-HER2 antibodies, anti-HER2 complementary bispecific antibodies) contained in the carrier are considered to be isolated.
  • Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • engineering includes any manipulation of the polypeptide or protein backbone, or post-translational modification of naturally occurring or recombinant proteins or polypeptides. Engineering includes mutations in the amino acid sequence, modifications of glycosylation or individual amino acid side chain groups, and combinations of these methods.
  • polypeptide refers to a polymer of amino acids and their equivalents, rather than the specific length of the product; therefore, “peptide” and “protein” are included in the definition of polypeptide.
  • the definition of polypeptide also includes “antibodies” defined in the present invention.
  • Dual variable domain (DVD) immunoglobulin is a tetravalent molecule containing two different antigen binding domains by inserting the heavy chain variable region (VH) and light chain variable region (VL) domains of another antibody into the N-terminus of the heavy chain and light chain of a normal IgG antibody. Therefore, each half of DVD-Ig contains a heavy chain polypeptide and a light chain polypeptide, which form two independent antigen binding domains after dimerization, one of which is an Fv fragment and the other is a Fab fragment or an IgG domain. DVD-Ig can be bispecific, that is, it can bind to two different epitopes of an antigen, or bind to two different antigens.
  • expression vector refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted and the protein can be expressed.
  • the vector can be used to transform, transduce or transfect host cells so that the genetic material elements it carries can be expressed in the host cells.
  • host cell refers to a cell into which an exogenous polynucleotide or nucleic acid and/or vector is introduced.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without considering the number of generations. Offspring may not be completely identical to the parental cell in nucleic acid content and may contain mutations.
  • the present invention includes mutant offspring with the same function or biological activity that is screened or selected in the initially transformed cells.
  • the terms "subject”, “patient” or “individual” can be used interchangeably, including but not limited to: mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, dogs, cats, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, sheep or other non-human mammals; non-mammals, including, for example, non-mammalian vertebrates, such as birds (e.g., chickens, emus or ducks) or fish; and non-mammalian invertebrates.
  • the subjects and pharmaceutical compositions involved in the uses or methods of the present invention are used to (prophylactically and/or therapeutically) treat non-human animals.
  • treating or "to treat” for a disease or symptom means alleviating or "to alleviate” a disease or symptom, reducing the rate of onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.
  • therapeutically effective amount refers to a dose or concentration that is effective to prevent or improve symptoms associated with a disease or condition and/or reduce the severity of a disease or condition at a desired dose and for a desired period of time.
  • the therapeutically effective amount of the formulation, antibody, or antigen-binding fragment thereof, ADC, or composition of the present invention may vary according to a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion or ADC to stimulate a desired response in the individual.
  • a therapeutically effective amount may also be considered to be any toxic or harmful effect of the formulation, antibody, or antigen-binding fragment thereof, ADC, or composition that is inferior to a therapeutically beneficial effect.
  • effective amount refers to an amount of an active ingredient or agent sufficient to provide a clinical benefit to a subject (including but not limited to improving, alleviating, or alleviating a disease, condition, or its associated symptoms, delaying or stopping disease progression).
  • low level of HER2 refers to the expression of HER2 in paraffin-embedded tissue sections of tumor biopsies as determined by immunohistochemistry (IHC) (e.g.
  • HER2 negative or “HER2 expression negative” or “expressing limited levels of HER2” refers to cancer cells/tumors, subjects or patients that have low HER2 expression as described above and an IHC assay score of 0 (no staining observed or membranous staining present in less than 10% of tumor cells).
  • HER2 positive or “HER2 expression positive” or “HER2 overexpression” refers to cancer cells/tumors, subjects or patients whose IHC assay scores are IHC 3+ (strong intact membrane staining observed in more than 10% of tumor cells). HER2 positive also includes IHC assay scores of Cancer cells/tumors, subjects or patients that are IHC 2 + and scored positive in both FISH tests (eg, Subtractive Probe Technology Chromogenic In Situ Hybridization [SPoT-Light HER2 CISH] test, Inform Dual In Situ Hybridization [Inform HER2Dual ISH] test) (IHC 2 + /FISH + ).
  • the "bystander effect” or “bystander killing effect” of ADC is mediated by free membrane-permeable small molecule toxins (e.g., anticancer agents), which are released by target molecule-positive (e.g., HER2-positive) cancer cells and passively diffuse into the tumor microenvironment, thereby killing neighboring cells, including neighboring cancer cells that do not express or low-express the target molecule (e.g., HER2-negative) and are insensitive to ADC.
  • target molecule-positive e.g., HER2-positive cancer cells
  • the term "pharmaceutically acceptable” or “pharmaceutically acceptable” means that the referenced carriers, vehicles, diluents, excipients and/or salts are generally chemically and/or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.
  • the terms “comprises” or “includes” or “contains” or “has” or “involving” are used interchangeably, meaning that the elements, integers or steps described are included, but any other elements, integers or steps are not excluded.
  • the terms “comprises” or “includes” or “contains” or “has” or “involving” are used, unless otherwise specified, the situation consisting of the elements, integers or steps described is also covered.
  • the term “optionally” means that the object it modifies is present or absent, for example, "the kit comprises optionally at least one additional tumor therapeutic agent” means that the kit may or may not comprise at least one additional tumor therapeutic agent.
  • Anti-HER2 antibodies or antigen-binding fragments thereof are provided.
  • the present invention provides an isolated anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER2 antibody or the antigen-binding fragment thereof can specifically bind to the extracellular domain of HER2.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1, D3 and/or D4 of the HER2 extracellular region, preferably D1 or D3 of the HER2 extracellular region.
  • the anti-HER2 complementary bispecific antibody of the present invention can be constructed using the antigen-binding domains of two monospecific anti-HER2 antibodies, wherein the two monospecific antibodies can each bind to different epitopes of the HER2 extracellular region, and there is no competitive inhibition relationship between the two monospecific antibodies in the antigen competitive binding test (i.e., the antigen-binding epitopes do not overlap).
  • the antigen-binding domains of two monospecific anti-HER2 antibodies can be used to construct the anti-HER2 complementary bispecific antibodies of the present invention, wherein the two monospecific antibodies can simultaneously bind to different epitopes in the extracellular region of HER2. Further, the two monospecific antibodies can simultaneously bind to any two epitopes among D1, D3 and D4 in the extracellular region of HER2, preferably, can simultaneously bind to D1 and D3 in the extracellular region of HER2, for example, wherein the antigen-binding domain of the first anti-HER2 antibody binds to D3 in the extracellular region of HER2, and the antigen-binding domain of the second anti-HER2 antibody binds to D1 in the extracellular region of HER2.
  • the anti-HER2 antibody or its antigen-binding fragment has strong binding activity to tumor cells expressing HER2, including HER2-high-expressing tumor cells (e.g., breast cancer cells SKBR-3, breast ductal cancer cells BT474, gastric cancer cells NCI-N87, and ovarian cancer cells SKOV-3), tumor cells expressing moderate levels of HER2 (e.g., breast cancer cells JIMT-1), HER2-low-expressing tumor cells (e.g., human bladder cancer cells RT-112, breast cancer cells ZR-75-1 and T47D), and/or tumor cells expressing limited levels of HER2 (e.g., breast cancer cells MCF-7).
  • HER2-high-expressing tumor cells e.g., breast cancer cells SKBR-3, breast ductal cancer cells BT474, gastric cancer cells NCI-N87, and ovarian cancer cells SKOV-3
  • tumor cells expressing moderate levels of HER2 e.g., breast cancer cells JIMT-1
  • HER2-low-expressing tumor cells
  • the anti-HER2 antibody or its antigen-binding fragment has a binding affinity (K D ) value of ⁇ 5 ⁇ 10 -8 M, preferably ⁇ 1 ⁇ 10 -8 M, ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M, and more preferably ⁇ 1 ⁇ 10 -9 M for HER2.
  • the anti-HER2 antibody or antigen-binding fragment thereof does not cross-bind with other members of the ErbB/HER family (including EGFR, HER3 and HER4).
  • the anti-HER2 antibody or its antigen-binding fragment does not affect the regulation of HER2 expressed on the cell surface and its downstream signal transduction pathways.
  • 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.
  • the anti-HER2 antibody or its antigen-binding fragment does not induce, block, or inhibit HER2 dimerization (including HER2:HER4 dimerization and/or HER2:HER3 dimerization) that is dependent on or independent of ligand induction and its downstream signal transduction pathways.
  • HER2 dimerization including HER2:HER4 dimerization and/or HER2:HER3 dimerization
  • the ligands include NRG-1 or Heregulins.
  • the anti-HER2 antibody of the present invention may also optionally include F(ab')2, Fab, Fab', Fv, scFv, scFv-Fc, single domain antibody (sdAb), or have IgG type.
  • the antibody of the present invention may be a mouse antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody or an antibody fragment, as long as the antibody can specifically recognize the epitopes of D1, D3 and/or D4 (preferably D1 and/or D3) of the extracellular region of HER2, and has no effect on HER2 expressed on the surface of tumor cells and its signal transduction pathway.
  • the anti-HER2 antibody is selected from mouse anti-human HER2 antibodies and humanized antibodies and optimized antibodies thereof. In some embodiments, the anti-HER2 antibody comprises scFv, scFv-Fc, Fab fragment and/or has IgG type.
  • the antibody that specifically binds to D3 of the extracellular region of HER2 can be in the form of scFv or scFv-Fc, and the antibody that specifically binds to D1 of the extracellular region of HER2 can be in the form of Fab or IgG, wherein Fab and scFv can be converted at will, and the conversion method is known in the art (see, for example, the method described in Zhou et al., Mol Cancer Ther 2012, 11: 1167-1476).
  • the present invention provides an anti-HER2 antibody or an antigen-binding fragment thereof comprising the CDRs and/or variable regions of the antibodies shown in Table 2, which recognizes D3 of the extracellular region of HER2 and does not induce, block, or inhibit the phosphorylation and/or dephosphorylation of tyrosine residues in the intracellular domain of HER2, and/or does not induce, block, or inhibit the dimerization of HER2 and its downstream signal transduction pathways that are dependent on or independent of NRG-1 induction.
  • the present invention also provides an anti-HER2 antibody or an antigen-binding fragment thereof capable of recognizing D1 of the extracellular region of HER2, which does not induce, block, or inhibit the phosphorylation and/or dephosphorylation of tyrosine residues in the intracellular domain of HER2, and/or does not induce, block, or inhibit the dimerization of HER2 and its downstream signal transduction pathways that are dependent on or independent of NRG-1 induction.
  • the antibody comprises an anti-HER2 antibody or an antigen-binding fragment thereof comprising the CDRs and/or variable regions of the antibodies shown in Table 3.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention further comprises CDRs, variable regions, or light chains and heavy chains of the anti-HER2 antibody or antigen-binding fragment thereof that recognize the HER2 extracellular region D4.
  • the heavy chain variable region CDRs and light chain variable region CDRs of the anti-HER2 antibody of the present invention are defined by the Kabat numbering system.
  • the CDR region can also be defined based on other numbering systems/methods such as Chothia and IMGT, AbM or Contact of the heavy chain/light chain variable region sequence, and the CDR region defined by other numbering systems/methods and the CDR region defined by Kabat used in the present invention are within the scope of protection of the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment of the present invention can specifically bind to D3 of the extracellular region of HER2, which comprises a VH amino acid sequence as shown in SEQ ID NO:1 and/or a VL amino acid sequence as shown in SEQ ID NO:2.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises a VH amino acid sequence as shown in SEQ ID NO:3, 7, or 9 and/or a VL amino acid sequence as shown in SEQ ID NO:4, 8, or 10.
  • the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO:3 and/or a VL amino acid sequence as shown in SEQ ID NO:4.
  • the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO:7 and/or a VL amino acid sequence as shown in SEQ ID NO:8.
  • the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the extracellular region of HER2 of the present invention comprises a VH amino acid sequence as shown in SEQ ID NO:9 and/or a VL amino acid sequence as shown in SEQ ID NO:10.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D4 of the extracellular region of HER2, which comprises the VH amino acid sequence shown in SEQ ID NO:5 and/or the VL amino acid sequence shown in SEQ ID NO:6.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D3 in the extracellular region of HER2 of the present invention comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequences shown in SEQ ID NOs: 11, 14 and 32 or variants thereof, including humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D3 in the extracellular region of HER2 of the present invention further comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequences shown in SEQ ID NOs: 38, 46 and 49 or variants thereof, wherein the variants include humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:32, and LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:49.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D1 of the present invention that specifically binds to the extracellular region of HER2 comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO:3 or the amino acid sequences shown in SEQ ID NOs:98, 100 and 110 or variants thereof, one or more CDRs in the VH amino acid sequence shown in SEQ ID NO:7 or the amino acid sequences shown in SEQ ID NOs:205, 206 and 207 or variants thereof, and/or one or more CDRs in the VH amino acid sequence shown in SEQ ID NO:9 or the amino acid sequences shown in SEQ ID NOs:211, 212 and 213 or variants thereof, and the variants include humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to D1 of the present invention that is extracellular region of HER2 comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence as shown in SEQ ID NOs:114, 116 and 139 or a variant thereof, one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence as shown in SEQ ID NOs:208, 209 and 210 or a variant thereof, and/or one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence as shown in SEQ ID NOs:214, 215 and 216 or a variant thereof, wherein the variants include humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D1 in the extracellular region of HER2 of the present invention comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100 and HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116 and LCDR3 as shown in SEQ ID NO:139; or, HCDR1 as shown in SEQ ID NO:205, HCDR2 as shown in SEQ ID NO:206 and LCDR3 as shown in SEQ ID NO:210.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D4 in the extracellular region of HER2 of the present invention comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO:5 or the amino acid sequences shown in SEQ ID NOs:199, 200 and 201 or variants thereof, including humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D4 in the extracellular region of HER2 of the present invention comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:6 or the amino acid sequences shown in SEQ ID NOs:202, 203 and 204 or variants thereof, including humanized antibodies or any other variants described in the present invention.
  • the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D4 in the extracellular region of HER2 of the present invention comprises HCDR1 as shown in SEQ ID NO:199, HCDR2 as shown in SEQ ID NO:200, and HCDR3 as shown in SEQ ID NO:201, and LCDR1 as shown in SEQ ID NO:202, LCDR2 as shown in SEQ ID NO:203, and LCDR3 as shown in SEQ ID NO:204.
  • the VH and/or VL of the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can be used as a starting material for engineering transformation to prepare an antibody described herein that is more suitable for administration to the human body.
  • the antibody can be engineered by mutating one or more amino acid residues in one or two variable regions (i.e., VH and/or VL), for example, mutating one or more CDR regions, and/or one or more FR framework regions.
  • the anti-HER2 antibody variable region of the present invention is engineered by CDR transplantation (Grafting).
  • Antibodies mainly interact with the target antigen through the amino acid residues of the six CDRs of the heavy chain variable region and the light chain variable region. Therefore, the amino acid sequence in the CDR region between each antibody is more diverse than the sequence (e.g., FR) outside the CDR region.
  • recombinant antibodies can be expressed by constructing an expression vector to mimic the properties of a specific naturally occurring antibody, wherein the expression vector contains the CDR sequence from a specific naturally occurring antibody transplanted to 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; see also US5225539, US5530101, US5585089, US5693762, and US6180370).
  • the anti-HER2 antibodies of the present invention may also contain different framework region sequences.
  • framework region sequences can be obtained from public DNA databases or published references involving germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in In the "V Base" human germline sequence database (available at the website www.mrc-cpe.cam.ac.uk/vbase); and Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No.
  • germline DNA sequences of human heavy and light chain variable region genes can be found in the IMGT database, for example, the following heavy chain germline sequences found in human immunoglobulins can be obtained by IMGT accession number: IGHV3-23 (DP47; VH26; V3-23) or IGHV7-4. As another example, the following light chain germline sequences found in human immunoglobulins can be obtained by IMGT accession number: IGKV1-39 or IGKV1-39*01.
  • the antibody amino acid sequences can be compared based on a compiled protein sequence database using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al., Nucleic Acids Res 1997, 25:3389-3402).
  • the preferred framework sequence used in the anti-HER2 antibody of the present invention is a receptor framework region that is structurally similar (or highly homologous) to the murine parent antibody framework sequence of the present invention.
  • the CDR1 region sequence, CDR2 region sequence, and CDR3 region sequence of VH or VL can be transplanted to the receptor framework region, respectively, and the receptor framework region has a sequence that is identical to or has the highest homology to the germline immunoglobulin gene in which it is located.
  • the present invention selects to transplant the CDR regions of VH shown in SEQ ID NO:1 or 3 and VL shown in SEQ ID NO:2 or 4 into the human IgG FR region to obtain a humanized antibody, wherein the humanized antibody can not only maintain the antigen binding activity similar to that of the parent antibody comprising the amino acid sequence of VH shown in SEQ ID NO:1 and VL shown in SEQ ID NO:2, or the amino acid sequence of VH shown in SEQ ID NO:3 and VL shown in SEQ ID NO:4, but also does not affect the HER2 downstream signal transduction pathway, for example, does not affect the phosphorylation of tyrosine residues in the intracellular domain of HER2 (e.g., Y1248 phosphorylation) and/or the ligand (e.g., NRG-1 or Heregulins)-induced HER2 dimerization and its mediated downstream signal transduction pathway.
  • the humanized antibody can not only maintain the antigen binding activity similar to that of the parent antibody comprising the
  • VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of other anti-HER2 antibodies that bind to HER2 can also be "mixed and matched" with the VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of the anti-HER2 antibodies of the present invention.
  • VH and VL chains or the CDRs within these chains, or full-length heavy chain and full-length light chain sequences
  • the VH sequence from a specific VH/VL pair is replaced by a structurally similar VH sequence.
  • the VL sequence from a specific VH/VL pair is replaced by a structurally similar VL sequence.
  • the full-length heavy chain sequence from a specific full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence.
  • the full-length light chain sequence from a specific full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.
  • the antibody or antigen-binding fragment thereof of the present invention 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 VL of another anti-HER2 antibody, wherein the antibody specifically binds to D3 or D1 of the extracellular region of HER2.
  • the antibody or antigen-binding fragment thereof of the present invention comprises: (a) a heavy chain variable region comprising an amino acid sequence listed in Table 2 or Table 3, or VH of another anti-HER2 antibody, wherein the antibody specifically binds to D3 or D1 of the extracellular region of HER2; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2 or Table 3.
  • the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 52.
  • the antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.
  • the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 81.
  • the antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.
  • the humanized antibody of the present invention comprises a VH 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 sequence shown in SEQ ID NO: 52, and a VL 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 sequence shown in SEQ ID NO: 81.
  • the antibody or antigen-binding fragment thereof specifically binds to D3 of the extracellular region of HER2.
  • the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VH amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 143 or 170.
  • the antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2.
  • the humanized antibody or antigen-binding fragment thereof of the present invention comprises a VL amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 171.
  • the antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2.
  • the humanized antibody of the present invention comprises a VH 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 sequence shown in SEQ ID NO: 143 or 170, and a VL 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 sequence shown in SEQ ID NO: 171.
  • the antibody or antigen-binding fragment thereof specifically binds to D1 of the extracellular region of HER2.
  • the CDR sequence can be transplanted to a framework region containing one or more mutations compared to the germline sequence, for example, mutating the amino acid residues in the framework region can maintain or enhance the antigen binding ability of the antibody (see, for example, US5530101, US5585089, US5693762, and US6180370).
  • the CDR of the parent antibody of the present invention is transplanted to a receptor framework region having one or more mutations compared to the germline sequence, which can increase the degree of humanization of the anti-HER2 antibody of the present invention and/or improve antigen binding affinity.
  • the FR region of the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 of the present invention has one or more amino acid mutations to improve the antigen-binding affinity of the humanized antibody.
  • one or more amino acid mutations are made to the FR region of the VH shown in SEQ ID NO:52.
  • amino acid mutations can be made in HFR1, for example, an amino acid mutation is made to the serine residue at position 30 of HFR1 (corresponding to position 30 of the VH amino acid sequence shown in SEQ ID NO:52); and/or SEQ ID NO:53 is mutated to the serine residue at position 30 of HFR1;
  • One or more amino acid mutations are made in the FR region of the VL shown in D NO:81.
  • one or more amino acid mutations can be made in LFR1, LFR2 and/or LFR3, specifically, one or more amino acid mutations are made in the aspartic acid residue at position 2, the methionine residue at position 4 of LFR1, the alanine residue at position 9 of LFR2 (corresponding to position 43 of the VL amino acid sequence shown in SEQ ID NO:81), and the threonine residue at position 29 of LFR3 (corresponding to position 85 of the VL amino acid sequence shown in SEQ ID NO:81).
  • the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 of the present invention has an amino acid mutation S30N in the HFR region relative to the VH amino acid sequence shown in SEQ ID NO:52, and/or it has one or more amino acid mutations, including D2I, M4L, A43S, and T85V, in the LFR region relative to the VL amino acid sequence shown in SEQ ID NO:81, which can significantly improve the antigen binding affinity of the antibody.
  • the FR region of the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 of the present invention has one or more amino acid mutations to improve the degree of humanization of the antibody of the present invention.
  • one or more amino acid mutations are made in the FR region of the VH shown in SEQ ID NO:52.
  • amino acid mutations can be made in HFR1 and/or HFR2.
  • amino acid mutations can be made in the tyrosine residue at position 29 of HFR1 and/or the alanine residue at position 14 of HFR2 (corresponding to position 49 of the amino acid sequence shown in SEQ ID NO:52); and/or the FR of the VL shown in SEQ ID NO:81 is mutated.
  • One or more amino acid mutations are made in the HFR region.
  • amino acid mutations can be made in LFR3, for example, an amino acid mutation is made to the phenylalanine residue at position 31 of LFR3 (corresponding to position 87 of the VL amino acid sequence shown in SEQ ID NO:81).
  • the anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 has the following amino acid mutations Y29F and/or A49S in HFR relative to the VH amino acid sequence shown in SEQ ID NO:52, and/or it has an amino acid mutation F87Y in the LFR region relative to the VL amino acid sequence shown in SEQ ID NO:81, which can improve the degree of humanization of the antibody.
  • the FR region of the anti-HER2 humanized antibody D1 that specifically binds to the extracellular region of HER2 of the present invention has one or more amino acid mutations.
  • one or more amino acid mutations are made in the FR region of the VH shown in SEQ ID NO: 143.
  • One or more amino acid mutations can be made in HFR1 and/or HFR3 to improve the degree of humanization of the anti-HER2 antibody of the present invention.
  • one or more amino acid mutations are performed at the serine residue at position 9, the valine residue at position 18 of HFR1, and/or the valine residue at position 3 of HFR3 (corresponding to position 68 of the VH amino acid sequence shown in SEQ ID NO: 143), the phenylalanine residue at position 4 (corresponding to position 69 of the VH amino acid sequence shown in SEQ ID NO: 143), the leucine residue at position 6 (corresponding to position 71 of the VH amino acid sequence shown in SEQ ID NO: 143), the valine residue at position 10 (corresponding to position 75 of the VH amino acid sequence shown in SEQ ID NO: 143), the isoleucine residue at position 17 (corresponding to position 82 of the VH amino acid sequence shown in SEQ ID NO: 143), and the phenylalanine residue at position 29 (corresponding to position 91 of the VH amino acid sequence shown in SEQ ID NO: 143).
  • One or more amino acid mutations may also be made in the FR region of the VL shown in SEQ ID NO: 171 to improve the degree of humanization of the anti-HER2 antibody of the present invention.
  • an amino acid mutation may be made in the asparagine residue at position 15 of LFR2 (corresponding to position 49 of the VL amino acid sequence shown in SEQ ID NO: 171).
  • the anti-HER2 humanized antibody D1 of the present invention that specifically binds to the extracellular region of HER2 has one or more of the following amino acid mutations in the HFR region relative to the VH amino acid sequence shown in SEQ ID NO: 143: S9G, V18L, V68T or V68S, F69I, L71V or L71R, V75K or V75T, I82L or I82M, F91Y.
  • the humanized antibody D1 that specifically binds to the extracellular region of HER2 comprises one or more of the following amino acid mutations in the HFR region relative to the VH amino acid sequence shown in SEQ ID NO: 143: V68T, F69I, L71R, V75K and F91Y, which can significantly improve the degree of humanization of the antibody.
  • the present invention performs amino acid mutations in the CDR regions of the VH and/or VL of a humanized antibody to improve one or more properties of the antibody, for example, to increase the degree of humanization, improve antigen binding affinity, increase expression, and enhance stability (e.g., reduce the potential risk of aspartic acid isomerization and/or asparagine deamidation).
  • Site-directed mutagenesis or PCR-induced mutations can be performed, and the effects of the mutations on the functional properties of the antibody can be evaluated by in vitro or in vivo detection methods known in the art.
  • the mutations can be amino acid substitutions, additions or deletions, preferably amino acid substitutions. In particular, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues are mutated in the heavy chain CDR or light chain CDR regions.
  • the anti-HER2 humanized antibody of the present invention that specifically binds to D3 in the extracellular region of HER2 has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations in the heavy chain CDR region or the light chain CDR region.
  • one or more amino acid mutations are made in the CDR region of the VH shown in SEQ ID NO: 52, and further, one, two, three, four, or five amino acid mutations may be made in HCDR1, HCDR2, and/or HCDR3.
  • One or more amino acid mutations are performed at the serine residue at position 1 of HCDR1 (corresponding to position 31 of VH shown in SEQ ID NO: 52), at the glycine residue at position 5 of HCDR2 (corresponding to position 53 of VH shown in SEQ ID NO: 52), at the serine residue at position 7 (corresponding to position 55 of VH shown in SEQ ID NO: 52), at the threonine residue at position 9 (corresponding to position 57 of VH shown in SEQ ID NO: 52), at the proline residue at position 12 (corresponding to position 60 of VH shown in SEQ ID NO: 52), at the aspartic acid residue at position 13 (corresponding to position 61 of VH shown in SEQ ID NO: 52), at the serine residue at position 14 (corresponding to position 62 of VH shown in SEQ ID NO: 52), and/or at the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH shown in SEQ ID NO: 52).
  • one or more amino acid mutations are performed on HCDR1, HCDR2 and/or HCDR3 in the VH shown in SEQ ID NO:52, for example, amino acid mutations are performed on the serine residue at position 7 of HCDR2 of the VH shown in SEQ ID NO:52 (corresponding to position 55 of VH shown in SEQ ID NO:52), the threonine residue at position 9 (corresponding to position 57 of VH shown in SEQ ID NO:52), the proline residue at position 12 (corresponding to position 60 of VH shown in SEQ ID NO:52), the aspartic acid residue at position 13 (corresponding to position 61 of VH shown in SEQ ID NO:52), the serine residue at position 14 (corresponding to position 62 of VH shown in SEQ ID NO:52), and/or the alanine residue at position 3 (corresponding to position 97 of VH shown in SEQ ID NO:52).
  • one or more amino acid mutations are made in the CDR region of the VL shown in SEQ ID NO: 81, and further, one, two, or three amino acid mutations can be made in LCDR1, LCDR3, and/or LCDR3.
  • one or more amino acid mutations can be made in the valine residue at position 7 of LCDR1 (corresponding to position 27c of VL shown in SEQ ID NO: 81), the histidine residue at position 8 (corresponding to position 27d of VL shown in SEQ ID NO: 81), the glycine residue at position 11 (corresponding to position 29 of VL shown in SEQ ID NO: 85), the phenylalanine residue at position 6 of LCDR2 (corresponding to position 55 of VL shown in SEQ ID NO: 81), and/or in the serine residue at position 1 (corresponding to position 89 of VL shown in SEQ ID NO: 81), the tyrosine residue at position 8 (corresponding to position 96 of VL shown in SEQ ID NO: 81).
  • the present invention performs multiple amino acid mutations on the CDR regions of VH and VL of the above-mentioned anti-HER2 humanized antibody D3 that specifically binds to the extracellular region of HER2 to improve the degree of humanization of the antibody of the present invention, increase the expression level, and/or improve the stability (for example, reduce the potential risk of aspartic acid isomerization).
  • one or more amino acid mutations are made to the serine residue at position 7 of HCDR2 of VH shown in SEQ ID NO:52 (corresponding to position 55 of VH shown in SEQ ID NO:52), the threonine residue at position 9 (corresponding to position 57 of VH shown in SEQ ID NO:52), the proline residue at position 12 (corresponding to position 60 of VH shown in SEQ ID NO:52), the aspartic acid residue at position 13 (corresponding to position 61 of VH shown in SEQ ID NO:52), the serine residue at position 14 (corresponding to position 62 of VH shown in SEQ ID NO:52), and the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH shown in SEQ ID NO:52), and the glycine residue at position 11 of LCDR1 of VL shown in SEQ ID NO:81 (corresponding to position 29 of VL shown in SEQ ID NO:81) is subjected to an amino acid mutation.
  • any one or two of the proline residue at position 12 of HCDR2 of VH shown in SEQ ID NO:52 are selected for amino acid mutation, and the alanine residue at position 3 of HCDR3 (corresponding to position 97 of VH shown in SEQ ID NO:52), and the glycine residue at position 11 of LCDR1 of VL shown in SEQ ID NO:81 (corresponding to position 29 of VL shown in SEQ ID NO:81) are selected for amino acid mutation.
  • the anti-HER2 humanized antibody of the present invention that specifically binds to D1 in the extracellular region of HER2 has no more than 1, 2, 3, 4, or 5 amino acid mutations in the heavy chain CDR region or the light chain CDR region.
  • one or more amino acid mutations are performed in the CDR region of the VH shown in SEQ ID NO:143. Furthermore, one, two, three, four, or five amino acid mutations may be performed in HCDR1, HCDR2 and/or HCDR3.
  • one or more amino acid mutations may be made at the serine residue at position 3 of HCDR1 (corresponding to position 33 of VH shown in SEQ ID NO: 143), at the glutamate residue at position 5 (corresponding to position 53 of VH shown in SEQ ID NO: 143), at the glutamate residue at position 8 (corresponding to position 56 of VH shown in SEQ ID NO: 143), at the aspartic acid residue at position 14 (corresponding to position 62 of VH shown in SEQ ID NO: 143), at the phenylalanine residue at position 15 (corresponding to position 63 of VH shown in SEQ ID NO: 143), and/or at the arginine residue at position 3 (corresponding to position 97 of VH shown in SEQ ID NO: 143), at the tyrosine residue at position 4 (corresponding to position 98 of VH shown in SEQ ID NO: 143), at the aspartic acid residue at position 5 (corresponding to position 99 of VH shown in SEQ ID NO: 143) of HCDR2.
  • one or more amino acid mutations are made in the CDR region of the VL shown in SEQ ID NO: 171, for example, one or more amino acid mutations can be made in the lysine residue at position 1 of LCDR1 (corresponding to position 24 of VL shown in SEQ ID NO: 171), in the serine residue at position 1 of LCDR2 (corresponding to position 50 of VL shown in SEQ ID NO: 171), in the tyrosine residue at position 4 (corresponding to position 53 of VL shown in SEQ ID NO: 171), in the tyrosine residue at position 6 (corresponding to position 55 of VL shown in SEQ ID NO: 171), and/or in the histidine residue at position 3 of LCDR3 (corresponding to position 91 of VL shown in SEQ ID NO: 171).
  • the present invention performs multiple amino acid mutations on the CDR regions of the VH and VL of the above-mentioned anti-HER2 humanized antibody that specifically binds to the D1 of the HER2 extracellular region to improve the degree of humanization of the antibody of the present invention, enhance antigen binding affinity, increase expression, and/or improve stability (e.g., reduce the risk of potential asparagine deamidation).
  • the glutamic acid residue at position 5 of HCDR2 of VH shown in SEQ ID NO: 143 correspond to position 53 of VH shown in SEQ ID NO: 143
  • the lysine residue at position 1 of LCDR1 of VL shown in SEQ ID NO: 171 correspond to position 24 of VL shown in SEQ ID NO: 171
  • the serine residue at position 1 of LCDR2 Multiple amino acid mutations are performed in the following: an acid residue at position (corresponding to position 50 of VL as shown in SEQ ID NO: 171), a tyrosine residue at position 4 (corresponding to position 53 of VL as shown in SEQ ID NO: 171), a tyrosine residue at position 6 (corresponding to position 55 of VL as shown in SEQ ID NO: 171) and a histidine residue at position 3 of LCDR3 (corresponding to position 91 of VL as shown in SEQ ID NO: 171).
  • multiple amino acid mutations may be made to the CDR and framework regions of the variable regions of the humanized antibodies to further improve one or more properties of the antibody (e.g., increasing the degree of humanization, increasing antigen binding affinity, enhancing stability, and increasing expression levels).
  • multiple amino acid mutations are performed on one or two CDR regions and one HFR region of the VH shown in SEQ ID NO: 143 of the humanized antibody that specifically binds to the extracellular region of HER2, and/or the three CDR regions of the VL shown in SEQ ID NO: 171.
  • the glutamic acid residue at position 5 of HCDR2 of the VH shown in SEQ ID NO: 143 correspond to position 53 of the VH shown in SEQ ID NO: 143
  • the valine residue at position 3 of HFR3 corresponding to position 68 of the amino acid sequence of the VH shown in SEQ ID NO: 143
  • the phenylalanine residue at position 4 corresponding to position 69 of the amino acid sequence of the VH shown in SEQ ID NO: 143
  • the leucine residue at position 6 correspond to position 71 of the amino acid sequence of the VH shown in SEQ ID NO: 143
  • the valine residue at position 10 correspond to position 75 of the amino acid sequence of the VH shown in SEQ ID NO: 143
  • the phenylalanine residue at position 29 correspond to position 68 ...
  • the above-mentioned antibody or antigen-binding fragment of D3 that specifically binds to the extracellular region of HER2 comprises: HCDR1 as shown in SEQ ID NO: 11, 12, or 13; HCDR2 as shown in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31; and HCDR3 as shown in SEQ ID NO: 32, 33, 34, 35, 36, or 37; and LCDR1 as shown in SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 45; LCDR2 as shown in SEQ ID NO: 46, 47, or 48; and LCDR3 as shown in SEQ ID NO: 49, 50, or 51.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention specifically binds to D3 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively:
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:32
  • LCDR1 as shown in SEQ ID NO:38
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:49
  • HCDR1 as shown in SEQ ID NO:12 or 13
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:32
  • LCDR1 as shown in SEQ ID NO:38
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:49;
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:32
  • LCDR1 as shown in SEQ ID NO:38
  • LCDR2 as shown in SEQ ID NO:47 or 48
  • LCDR3 as shown in SEQ ID NO:49;
  • HCDR1 as shown in SEQ ID NO: 11
  • HCDR2 as shown in SEQ ID NO: 14
  • HCDR3 as shown in SEQ ID NO: 32
  • LCDR1 as shown in SEQ ID NO: 39, 40, 41, 42, 43, 44, or 45
  • LCDR1 as shown in SEQ ID NO: 46
  • LCDR2 LCDR3 as shown in SEQ ID NO:49; or
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:32
  • LCDR1 as shown in SEQ ID NO:38
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:50 or 51;
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:33, 34, 35, 36, or 37
  • LCDR1 as shown in SEQ ID NO:38
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:49
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:14
  • HCDR3 as shown in SEQ ID NO:37
  • LCDR1 as shown in SEQ ID NO:42
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:49;
  • HCDR1 as shown in SEQ ID NO:11
  • HCDR2 as shown in SEQ ID NO:21
  • HCDR3 as shown in SEQ ID NO:32
  • LCDR1 as shown in SEQ ID NO:42
  • LCDR2 as shown in SEQ ID NO:46
  • LCDR3 as shown in SEQ ID NO:49.
  • the anti-HER2 antibody or its antigen-binding fragment of the present invention specifically binds to D3 of the extracellular region of HER2, comprising: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:14, 17, 19, 21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:32 or 37, and HCDR4 as shown in SEQ ID NO:38 or 42.
  • LCDR1 as shown in SEQ ID NO:46
  • LCDR2 as shown in SEQ ID NO:49
  • amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the antibody or antigen-binding fragment thereof comprises: a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 14, 17, 19, 24, 29, 30, or 31, a HCDR3 as shown in SEQ ID NO: 32 or 37, and a LCDR1 as shown in SEQ ID NO: 38 or 42, a LCDR2 as shown in SEQ ID NO: 46, a LCDR3 as shown in SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:24, 29, 30, or 31 and HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46 and LCDR3 as shown in SEQ ID NO:49, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the antibody or antigen-binding fragment thereof not only has a high affinity for HER2 (for example, a KD value of ⁇ 1 ⁇ 10-8 M, or ⁇ 5 ⁇ 10-9 M, or ⁇ 1 ⁇ 10-9 M, preferably, a KD value of ⁇ 5 ⁇ 10-9 M, or ⁇ 1 ⁇ 10-9 M), but also has a higher degree of humanization and stability.
  • HER2 for example, a KD value of ⁇ 1 ⁇ 10-8 M, or ⁇ 5 ⁇ 10-9 M, or ⁇ 1 ⁇ 10-9 M, preferably, a KD value of ⁇ 5 ⁇ 10-9 M, or ⁇ 1 ⁇ 10-9 M
  • the above-mentioned antibody or antigen-binding fragment of D1 that specifically binds to the extracellular region of HER2 comprises: HCDR1 as shown in SEQ ID NO: 98 or 99; HCDR2 as shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109; HCDR3 as shown in SEQ ID NO: 110, 111, 112, or 113; and HCDR4 as shown in SEQ ID NO: 114.
  • LCDR1 as shown in SEQ ID NO:114 or 115
  • LCDR2 as shown in SEQ ID NO:116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138
  • LCDR3 as shown in SEQ ID NO:139, 140, 141, or 142.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention specifically binds to D1 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively:
  • HCDR1 as set forth in SEQ ID NO:98
  • HCDR2 as set forth in SEQ ID NO:100
  • HCDR3 as set forth in SEQ ID NO:110
  • LCDR1 as set forth in SEQ ID NO:114
  • LCDR2 as set forth in SEQ ID NO:116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, or 128, LCDR3 as set forth in SEQ ID NO:139; or
  • HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, LCDR3 as shown in SEQ ID NO:140, 141, or 142; or
  • HCDR1 as shown in SEQ ID NO:98
  • HCDR2 as shown in SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, or 109
  • HCDR3 as shown in SEQ ID NO:110
  • LCDR1 as shown in SEQ ID NO:114
  • LCDR2 as shown in SEQ ID NO:116
  • LCDR3 as shown in SEQ ID NO:139;
  • HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100, HCDR3 as shown in SEQ ID NO:111, 112, or 113, LCDR1 as shown in SEQ ID NO:114, LCDR2 as shown in SEQ ID NO:116, LCDR3 as shown in SEQ ID NO:139; or
  • HCDR1 as shown in SEQ ID NO:98
  • HCDR2 as shown in SEQ ID NO:100
  • HCDR3 as shown in SEQ ID NO:110
  • LCDR1 as shown in SEQ ID NO:115
  • LCDR2 as shown in SEQ ID NO:116
  • LCDR3 as shown in SEQ ID NO:139
  • HCDR1 as shown in SEQ ID NO:99
  • HCDR2 as shown in SEQ ID NO:100
  • HCDR3 as shown in SEQ ID NO:110
  • LCDR1 as shown in SEQ ID NO:114
  • LCDR2 as shown in SEQ ID NO:116
  • LCDR3 as shown in SEQ ID NO:139
  • HCDR1 as shown in SEQ ID NO:98
  • HCDR2 as shown in SEQ ID NO:102
  • HCDR3 as shown in SEQ ID NO:110
  • LCDR1 as shown in SEQ ID NO:115
  • LCDR2 as shown in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138
  • LCDR3 as shown in SEQ ID NO:142.
  • the anti-HER2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises the following HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:100 or 102, HCDR3 as shown in SEQ ID NO:110, LCDR1 as shown in SEQ ID NO:114 or 115, LCDR2 as shown in NO:116, 123, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138
  • the antibody or antigen-binding fragment thereof comprises: a HCDR1 as shown in SEQ ID NO:98, a HCDR2 as shown in SEQ ID NO:102, a HCDR3 as shown in SEQ ID NO:110, a LCDR1 as shown in SEQ ID NO:115, a LCDR2 as shown in SEQ ID NO:129, 133, 136, or 138, a LCDR3 as shown in SEQ ID NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the antibody or antigen-binding fragment thereof has a binding affinity (K D ) value for HER2 of ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M, preferably ⁇ 1 ⁇ 10 -9 M. More preferably, the antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102 and HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133 and LCDR3 as shown in SEQ ID NO:142, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the anti-HER2 antibody or antigen-binding fragment thereof 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.
  • the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to D3 of the extracellular region of HER2, which comprises: a polypeptide having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 100%, or at least 100% affinity) to the amino acid sequence shown in SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.
  • a polypeptide having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 100%, or at least 100% affinity) to the amino acid sequence shown in SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
  • the invention further comprises a heavy chain variable region VH that is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, and a light chain variable region VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence shown in SEQ ID NO:81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97.
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to D3 of the extracellular region of HER2, which comprises a heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and a light chain variable region having the LCDR1, LCDR2 and LCDR3, wherein the heavy chain variable region and the light chain variable region each comprise 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: 52 and the VL shown in SEQ ID NO: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, respectively; or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the antibody or antigen-binding fragment thereof that specifically binds to D3 in the extracellular region of HER2 comprises: an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in any one of SEQ ID NOs: 52, 57, 62, 68, 73, 78, 79 and 80, and the VL shown in SEQ ID NO: 81 or 92.
  • the antibody or its antigen-binding fragment comprises: an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 73, 78, 79, or 80 and the VL shown in SEQ ID NO: 92, respectively, and more preferably, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in SEQ ID NO: 80 and the VL shown in SEQ ID NO: 92, respectively.
  • the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises: a polypeptide having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) affinity to the amino acid sequence shown in SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170.
  • the invention further comprises a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence shown in SEQ ID NO:171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198.
  • VH heavy chain variable region having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence shown in SEQ ID NO:171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to D1 of the extracellular region of HER2, which comprises a heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and a light chain variable region having the LCDR1, LCDR2 and LCDR3, which comprises VH shown in SEQ ID NO: 143 and VH shown in SEQ ID NO: 171, 172, 173, 174, 175, 176, 177, 178, respectively.
  • the antibody or antigen-binding fragment thereof that specifically binds to D1 in the extracellular region of HER2 comprises: an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH shown in any one of SEQ ID NOs: 143, 148, 156, 158, 160, 161, 165 and 169, and the 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.
  • the antibody or antigen-binding fragment thereof comprises: a VH shown in SEQ ID NO: 169 and a VL shown in SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198 that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% affinity to the VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, respectively.
  • the present invention further 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 VH shown in SEQ ID NO: 169 and the VL shown in SEQ ID NO: 189, 193, 196, or 198, respectively.
  • the above-mentioned antibody or antigen-binding fragment thereof of the present invention comprises: the VH shown in SEQ ID NO: 169, and the VL shown in SEQ ID NO: 193.
  • the anti-HER2 humanized antibody or its antigen-binding fragment of the present invention has strong binding activity with cells expressing HER2, has no effect on the HER2-mediated signal transduction pathway, does not cross-bind with other members of the human ErbB/HER family (including EGFR, HER3 and HER4), and has a high degree of humanization and stability.
  • the present invention provides an anti-HER2 complementary bispecific antibody, the complementary bispecific antibody comprising two antigen binding domains capable of simultaneously and non-competitively binding to the HER2 extracellular region, wherein the first antigen binding domain and the second antigen binding domain each specifically bind to different epitopes of the HER2 extracellular region.
  • the complementary bispecific antibody can simultaneously bind to any two epitopes of D1, D3 and D4 of the HER2 extracellular region, preferably, can simultaneously bind to D1 and D3 of the HER2 extracellular region, for example, wherein the first antigen binding domain specifically binds to D3 of the HER2 extracellular region, and the second antigen binding domain specifically binds to D1 of the HER2 extracellular region, and the binding epitopes of the first and second antigen binding domains are different from the antigen binding epitopes of Trastuzumab or Pertuzumab.
  • the anti-HER2 complementary bispecific antibody of the present invention has the following functional characteristics:
  • the anti-HER2 complementary bispecific antibody is a tetravalent molecule that can simultaneously bind to two different epitopes in the extracellular region of HER2, thereby being able to cross-link HER2 on the cell surface to form antigen-antibody cross-linked aggregates or clusters.
  • the efficiency and size of the aggregate or cluster formation are subject to the abundance of the target antigen on the cell surface, and the formation of the cluster will trigger the cell's endocytosis reaction to it, and the rate and intensity of the endocytosis reaction are positively correlated with the size of the cluster.
  • the complementary bispecific antibody has a significantly enhanced internalization efficiency, and its endocytosis rate in HER2-overexpressing tumor cells is as high as 80% or more, and its endocytosis rate in HER2-low-expressing tumor cells is as high as 60% or more.
  • the complementary bispecific antibody forms clusters with HER2 on the cell surface and is internalized, and lysosomal trafficking is the dominant intracellular transport pathway. Therefore, the clusters formed by the complementary bispecific antibody and HER2 can be efficiently transported into lysosomes for degradation.
  • the complementary bispecific antibody can effectively induce the degradation of HER2 on the surface of tumor cells, thereby significantly inhibiting the proliferation of HER2-overexpressing tumor cells.
  • the complementary bispecific antibody does not affect the regulation of HER2 and its mediated downstream signal transduction pathways, including not inducing, blocking, or inhibiting ligand (e.g., NRG-1)-induced HER2 dimerization and activation of its downstream signal transduction pathways. Therefore, the anti-HER2 complementary bispecific antibody does not affect the normal biological function and regulation of HER2 and its mediated downstream signal transduction pathways in normal tissues or cells (e.g., cardiomyocytes).
  • the anti-HER2 complementary bispecific antibody has high stability and expression level, as well as high monomer content (about 98% or more).
  • the complementary bispecific antibody formats include, but are not limited to, bispecific formats based on scFv or Diabody (e.g., scFv-scFv or scFv-Fab or Fab-scFv), IgG-scFv fusion proteins, DVD-Ig, Quadroma, Knob-into-hole, Common Light Chain, CrossMab, CrossFab, SEEDbody, Leucine Zipper, Duobody, IgG1/IgG2, Dual-acting Fab (DAF)-IgG and Mab 2 bispecific formats (see, e.g., Klein et al., mAbs 2012, 4:653-663 and references cited therein).
  • scFv-scFv or scFv-Fab or Fab-scFv IgG-scFv fusion proteins
  • DVD-Ig Quadroma
  • Knob-into-hole Common Light Chain
  • CrossMab CrossFab
  • SEEDbody Leu
  • the exemplary anti-HER2 complementary bispecific antibody of the present invention adopts the DVD-Ig format, which not only has a high binding activity to HER2-expressing tumor cells, but also exhibits a high cell internalization rate. In addition, it can also ensure the stability of the complementary bispecific antibody of the present invention (for example, a high monomer content of about 98% or more).
  • the anti-HER2 complementary bispecific antibody has strong binding activity with cells expressing HER2, and has no cross-binding activity with other members of the human ErbB/HER family (including EGFR, HER3 and HER4).
  • the anti-HER2 complementary bispecific antibody exhibits significantly enhanced internalization efficiency.
  • the complementary bispecific antibody has an endocytosis rate of up to 80% or more in tumor cells overexpressing HER2 (IHC 3 + or IHC 2 + /FISH + ) compared to the corresponding monospecific anti-HER2 antibody or its antigen-binding fragment and Trastuzumab.
  • the complementary bispecific antibody has an endocytosis rate of up to 60% in tumor cells underexpressing HER2 (IHC 2 + /FISH - or IHC 1 + ) compared to the corresponding monospecific anti-HER2 antibody or its antigen-binding fragment and Trastuzumab.
  • the anti-HER2 complementary bispecific antibody can effectively promote the degradation of HER2 in tumor cells.
  • the complementary bispecific antibody can induce the degradation of HER2 in HER2-overexpressing tumor cells (e.g., BT474 cells) by Western-blot method detection, while the monospecific anti-HER2 antibody or its antigen-binding fragment corresponding to the complementary bispecific antibody and Trastuzumab cannot induce the degradation of HER2 in the tumor cells.
  • the anti-HER2 complementary bispecific antibody can effectively reduce the expression abundance of HER2 on the surface of tumor cells, so the anti-HER2 complementary bispecific antibody can significantly inhibit the proliferation of HER2-overexpressing tumor cells.
  • the anti-HER2 complementary bispecific antibody has a significant inhibitory effect on the proliferation of HER2-overexpressing tumor cells (e.g., BT474 cells), and its inhibitory activity is comparable to that of Trastuzumab, while the monospecific anti-HER2 antibody or its antigen-binding fragment corresponding to the complementary bispecific antibody cannot inhibit the proliferation of tumor cells.
  • HER2-overexpressing tumor cells e.g., BT474 cells
  • the anti-HER2 complementary bispecific antibody has no effect on the HER2-mediated signal transduction pathway, and maintains the characteristic that the corresponding monospecific anti-HER2 antibody or its antigen-binding fragment does not affect the biological function of HER2, so it will not have an adverse effect on the normal biological function of HER2 in normal tissues or cells (e.g., cardiomyocytes).
  • the anti-HER2 complementary bispecific antibody does not induce, block, or inhibit the phosphorylation and/or dephosphorylation of AKT in the downstream signaling pathway induced by ligand (e.g., NRG-1) induced HER2 dimerization and mediated by it.
  • ligand e.g., NRG-1
  • the anti-HER2 complementary bispecific antibody has a very low risk of potentially inducing cardiac toxic side effects.
  • the first and second antigen-binding domains of the anti-HER2 complementary bispecific antibody of the present invention can be derived from any two monospecific anti-HER2 antibodies or antigen-binding fragments thereof that do not compete with each other for binding to HER2 of the present invention, or can be derived from currently known anti-HER2 antibodies or antigen-binding fragments thereof.
  • the exemplary anti-HER2 complementary bispecific antibodies of the present invention can be constructed based on D3 that specifically binds to the extracellular region of HER2 shown in Table 2 and the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to the extracellular region of HER2 shown in Table 3.
  • the exemplary anti-HER2 complementary bispecific antibodies of the present invention comprise two different antigen-binding domains, wherein the first antigen-binding domain comprises at least one CDR region and/or any variable region of the anti-HER2 antibodies or antigen-binding fragments thereof that specifically bind to the extracellular region of HER2 shown in Table 2.
  • the second antigen-binding domain comprises at least one CDR region and/or any variable region of any anti-HER2 antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of HER2 shown in Table 3.
  • the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and/or light chain variable region CDRs
  • the heavy chain variable region CDRs of the first antigen-binding domain comprise the amino acid sequence of any one, two or three CDR regions of the heavy chain variable region CDRs listed in Table 2, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 13
  • the heavy chain variable region CDRs of the second antigen-binding domain comprise the amino acid sequences of any one, two or three of the heavy chain variable region CDRs listed in Table 3, or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with HCDR1, HCDR2 and HCDR3, respectively.
  • the light chain variable region CDRs of the second antigen binding domain comprise the amino acid sequence of any one, two, or three CDR regions of the light chain variable region CDRs listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to LCDR1, LCDR2, and LCDR3, respectively.
  • the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2, and comprises: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46 and LCDR3 as shown in SEQ ID NO:49, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2, and comprises: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:24, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46 and LCDR3 as shown in SEQ ID NO:49, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2, and comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, and LCDR1 as shown in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138 and LCDR2 as shown in SEQ ID NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2, and comprises: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133 and LCDR3 as shown in SEQ ID NO:142, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody further comprises a heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and/or a light chain variable region having LCDR1, LCDR2 and LCDR3, the heavy chain variable region of the first antigen-binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto;
  • the light chain variable region of the first antigen-binding domain comprises an amino acid sequence of any one of the VL amino acid sequences listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.
  • the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 3, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto;
  • the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VH amino acid sequences listed in Table 3, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97
  • the light chain variable region of the second antigen binding domain comprises an amino acid sequence of any one of the VL amino acid sequences listed in Table 3, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto;
  • the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2, and comprises: VH as shown in SEQ ID NO: 70, 72, 73, 76, 77, 78, 79, or 80, and VH as shown in SEQ ID NO: 92, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.
  • the first antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the extracellular region of HER2, and comprises: VH as shown in SEQ ID NO:73, 78, 79, or 80, and VL as shown in SEQ ID NO:92, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.
  • the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2, and comprises: VH as shown in SEQ ID NO:169, and VL as shown in SEQ ID NO:189, 190, 191, 192, 193, 194, 195, 196, 197, or 198, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.
  • the second antigen binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the extracellular region of HER2, and comprises: VH as shown in SEQ ID NO:169, and VL as shown in SEQ ID NO:193, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL, respectively.
  • the anti-HER2 complementary bispecific 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 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:21, 23, 24, 27, 28, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46, and LCDR3 as shown in SEQ ID NO:47.
  • the second antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:129, 130, 131, 132, 133, 134, 135, 136, 137, or 138, and LCDR3 as shown in SEQ ID NO:142.
  • the anti-HER2 complementary bispecific antibody comprises a first and a second antigen binding domain, wherein the first antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:24, 29, 30, or 31, HCDR3 as shown in SEQ ID NO:37, and LCDR1 as shown in SEQ ID NO:42, LCDR2 as shown in SEQ ID NO:46.
  • the first antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9
  • the second antigen binding domain comprises the following heavy chain variable region CDRs and light chain variable region CDRs, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each of the CDRs, respectively: HCDR1 as shown in SEQ ID NO:98, HCDR2 as shown in SEQ ID NO:102, HCDR3 as shown in SEQ ID NO:110, and LCDR1 as shown in SEQ ID NO:115, LCDR2 as shown in SEQ ID NO:133 and LCDR3 as shown in SEQ ID NO:142.
  • the anti-HER2 complementary bispecific 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
  • the heavy chain variable region VH and the light chain variable region VL comprise the VH amino acid sequence shown in SEQ ID NO: 70, 72, 73, 76, 77, 78, 79, or 80
  • the VL amino acid sequence shown in SEQ ID NO: 92 or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%,
  • the anti-HER2 complementary bispecific 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
  • the heavy chain variable region VH and the light chain variable region VL comprise the VH amino acid sequence as shown in SEQ ID NO: 73, 78, 79, or 80
  • the VL amino acid sequence as shown in SEQ ID NO: 92 or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%
  • the exemplary anti-HER2 complementary bispecific antibodies of the present invention can also be based on the D1 that specifically binds to the extracellular region of HER2 described in the present invention. and D4 anti-HER2 antibody or antigen-binding fragment thereof is constructed.
  • the exemplary anti-HER2 complementary bispecific antibody of the present invention comprises a first and a second antigen-binding domain, and the first and the second antigen-binding domains can simultaneously bind to HER2 without competing with each other, wherein the first antigen-binding domain comprises a VH amino acid sequence shown in SEQ ID NO:7 and a VL amino acid sequence shown in SEQ ID NO:8, or the first antigen-binding domain comprises a VH amino acid sequence shown in SEQ ID NO:9 and a VL amino acid sequence shown in SEQ ID NO:10, or the first antigen-binding domain comprises any at least one CDR region and/or any variable region of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 3, and can specifically bind to
  • the first or second antigen binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and/or light chain variable region CDRs
  • the heavy chain variable region CDRs of the first antigen binding domain comprise one, two, or three CDRs in the VH amino acid sequence shown in SEQ ID NO:7 or the amino acid sequences shown in SEQ ID NOs:205, 206 and 207 or their variants, wherein the variants include humanized antibodies or any other variants
  • the light chain variable region CDRs of the first antigen binding domain comprise one, two, or three CDRs in the VL amino acid sequence shown in SEQ ID NO:8 or the amino acid sequences shown in SEQ ID NOs:208, 209 and 210
  • the heavy chain variable region CDRs of the first antigen-binding domain include one, two, or three CDRs in the VH amino acid sequence shown in SEQ ID NO:9 or
  • the amino acid sequence of the first antigen-binding domain comprises an amino acid sequence with 6%, 97%, 98%, 99%, or 100% identity;
  • the heavy chain variable region CDRs of the second antigen-binding domain comprise one, two, or three CDRs in the VH amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence as shown in SEQ ID NOs:199, 200 and 201, or a variant thereof, which includes a humanized antibody or any other variant;
  • the light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs in the VL amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence as shown in SEQ ID NOs:202, 203 and 204, or a variant thereof, which includes a humanized antibody or any other variant.
  • the exemplary anti-HER2 complementary bispecific antibody of the present invention can also be constructed based on the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D3 and D4 of the HER2 extracellular region described in the present invention.
  • the exemplary anti-HER2 complementary bispecific antibody of the present invention comprises a first and a second antigen-binding domain, and the first and the second antigen-binding domain can simultaneously bind to HER2 without competing with each other, wherein the first antigen-binding domain comprises any at least one CDR region and/or any variable region of the anti-HER2 antibody or its antigen-binding fragment that specifically binds to D3 of the HER2 extracellular region shown in Table 2; the second antigen-binding domain comprises the VH amino acid sequence shown in SEQ ID NO:5 and the VL amino acid sequence shown in SEQ ID NO:6, and can specifically bind to D4 of the HER2 extracellular region.
  • the first or second antigen-binding domain of the anti-HER2 complementary bispecific antibody comprises heavy chain variable region CDRs and/or light chain variable region CDRs
  • the heavy chain variable region CDRs of the first antigen-binding domain comprises the amino acid sequence of any one, two, or three CDR regions of the heavy chain variable region CDRs listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • the light chain variable region CDRs of the first antigen-binding domain comprises the amino acid sequence of any one, two, or three CDR regions of the light chain variable region CDRs listed in Table 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89% or 100% identity thereto.
  • the heavy chain variable region CDRs of the second antigen-binding domain comprise one, two, or three CDRs in the VH amino acid sequence shown in SEQ ID NO:5 or the amino acid sequences shown in SEQ ID NOs:199, 200 and 201, or variants thereof, including humanized antibodies or any other variants
  • the light chain variable region CDRs of the first antigen-binding domain comprise one, two, or three CDRs in the VL amino acid sequence shown in SEQ ID NO:6 or the amino acid sequences shown in SEQ ID NOs:202, 203 and 204, or variants thereof, including humanized antibodies or any other variants.
  • the exemplary anti-HER2 complementary bispecific antibody of the present invention can be constructed in the form of DVD-Ig (see US7612181), and the first or second antigen binding domain of the anti-HER2 complementary bispecific antibody can be in the form of Fv/domain, or in the form of Fab or IgG/domain, that is, if the first antigen binding domain is Fv, then the second antigen binding domain is Fab or IgG; if the second antigen binding domain is Fv, then the first antigen binding domain is Fab or IgG.
  • the Fv domain is fused to or operably connected to the Fab or IgG domain through a linker sequence, and the linker not only has low immunogenicity, but also can ensure the stability of the complementary bispecific antibody, preferably a flexible peptide as a linker.
  • linker refers to a compound that connects two compounds, such as two polypeptide molecules (including but not limited to unmodified or modified amino acids or amino acid sequences).
  • a linker can be composed of one or more linker molecules, or can include a linker molecule and at least one A spacer molecule designed to separate the linker molecule and the compound by a specific distance.
  • operably linked refers to the linkage of amino acid sequences, peptides or proteins with different functional properties, such as the linkage of an Fv domain to a Fab or IgG domain via a linker sequence as described herein.
  • the first antigen-binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D3 of the HER2 extracellular region and is an Fv domain
  • the first antigen-binding domain comprises the heavy chain variable region CDRs and light chain variable region CDRs, and/or the heavy chain variable region VH and light chain variable region VL of the anti-HER2 antibodies or antigen-binding fragments thereof shown in Table 2
  • the second antigen-binding domain of the anti-HER2 complementary bispecific antibody specifically binds to D1 of the HER2 extracellular region and is a Fab domain or an IgG domain
  • the second antigen-binding domain comprises the heavy chain variable region CDRs and light chain variable region CDRs, and/or the heavy chain variable region VH and light chain variable region VL of the anti-HER2 antibody or antigen-binding fragment thereof shown in Table 3
  • the C-terminus of the VH domain in the Fv domain is fused to or operably connected to the N-terminus of the VH domain in
  • the linker sequence includes different copy numbers of GGGGS (G 4 S) sequences, such as 1, 2, 3, 4, or 5 copies.
  • the copy number of the linker G 4 S connecting the VH domain in the Fv domain of the anti-HER2 complementary bispecific antibody to the VH domain in the Fab or IgG domain of the complementary bispecific antibody may be the same as or different from the copy number of the linker G 4 S connecting the VL domain in the Fv domain of the complementary bispecific antibody to the VL domain in the Fab or IgG domain of the complementary bispecific antibody.
  • the copy number of the linker G 4 S connecting the VH domain between the Fv domain and the Fab or IgG domain is different from the copy number of the linker G 4 S connecting the VL domain between the two, wherein the copy number of the linker connecting the VH domains of the two is preferably 1, and the copy number of the linker connecting the VL domains of the two is preferably 3, thereby effectively ensuring the structural stability of the anti-HER2 complementary bispecific antibody of the present invention and improving the endocytosis rate in HER2-expressing tumor cells.
  • the anti-HER2 complementary bispecific antibody comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises VH and VL of an anti-HER2 antibody or an antigen-binding fragment thereof shown in Table 2, and the second antigen-binding domain comprises VH and VL of an anti-HER2 antibody or an antigen-binding fragment thereof 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 connected to the N-terminus of the VH domain in the Fab or IgG domain via a linker (e.g., (G 4 S) n , n is an integer greater than 0, such as 1-3; preferably (G 4 S) 1 ), and the C-terminus of the VL domain in the Fv domain is connected to the N-terminus of the VL domain in the Fab or IgG domain via a linker (e
  • the anti-HER2 complementary bispecific antibody of the present invention may also comprise a constant region, which comprises an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant region of the present invention comprises natural and mutant protein forms of the Fc region of the human IgG heavy chain constant region, and also comprises a truncated form of a polypeptide containing a hinge region that promotes dimer formation.
  • the Fc region comprises the CH2 and CH3 domains of the antibody. Fusion proteins (and oligomers formed therefrom) comprising the Fc portion provide the advantage of easy affinity chromatography purification by protein A (Protein A) or protein G (Protein G), as well as extended serum half-life.
  • the preferred Fc region is derived from human IgG, including IgG1, IgG2, IgG3 and IgG4. In this article, the positions of specific amino acid residues in the Fc region are determined according to the EU numbering system.
  • Fc region of an antibody One function of the Fc region of an antibody is to produce "effector functions" with the immune system when the antibody binds to its target molecule, including the production of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • 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 the 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 binding of Fc to proteins of the complement system such as C1q (Ward et al., Ther Immunol 1995, 2:77-94).
  • the anti-HER2 complementary bispecific antibodies of the invention comprise an engineered IgG Fc region to reduce Fc-mediated effector function.
  • Exemplary antibodies with reduced effector function include an Fc region containing the following amino acid mutations:
  • the preferred engineered Fc region is a human IgG1 Fc with L234F/L235E/P331S (EU numbering system) amino acid substitutions, which can reduce the binding of the Fc region to one or more Fc ⁇ Rs and C1q (Oganesyan et al., Acta Crystallogr D Biol Crystallogr 2008, 64: 700-704; US5624821; US6194551).
  • the Fc ⁇ R protein family includes Fc ⁇ RI (also known as CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; Fc ⁇ RII (also known as CD32), including isoforms Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIc; and Fc ⁇ RIII (also known as CD16), Including isoforms Fc ⁇ RIIIa and Fc ⁇ RIIIb (Jefferis et al., Immunol Lett 2002, 82: 57-65). Among them, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc and Fc ⁇ RIIIa can induce ADCC, endocytosis, phagocytosis and/or cytokine release.
  • Binding properties include but are not limited to binding specificity, binding affinity ( KD ), and dissociation and association rates ( kdis and ka, respectively). Those skilled in the art can analyze whether the engineered Fc region has altered ADCC and/or CDC activity by any one or more binding properties.
  • the heavy chain constant region and light chain constant region in the anti-HER2 complementary bispecific antibody of the present invention may be a heavy chain constant region from human IgG1 or IgG4 (which may be natural or mutated, such as the wild-type human IgG1 constant region shown in SEQ ID NO: 231), and a human Kappa ( ⁇ ) chain or Lambda ( ⁇ ) constant region.
  • the heavy chain constant region of the anti-HER2 complementary bispecific antibody comprises a human IgG1 Fc (as shown in SEQ ID NO: 232) with L234F/L235E/P331S (EU numbering system) amino acid substitutions, which has reduced binding affinity to one or more Fc ⁇ Rs (e.g., Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, and Fc ⁇ RIIIa) and C1q.
  • Fc ⁇ Rs e.g., Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, and Fc ⁇ RIIIa
  • the L234F/L235E/P331S mutation is introduced into the heavy chain constant region of the anti-HER2 complementary bispecific antibody so that the complementary bispecific antibody does not substantially bind to Fc ⁇ RI, Fc ⁇ RIIa (167H), Fc ⁇ RIIb, Fc ⁇ RIIIa (176V), and/or Fc ⁇ RIIIa (176F).
  • the introduction of L234F/L235E/P331S mutations in the heavy chain constant region of the anti-HER2 complementary bispecific antibody makes the complementary bispecific antibody substantially non-binding to C1q.
  • the introduction of L234F/L235E/P331S mutations in the heavy chain constant region of the anti-HER2 complementary bispecific antibody does not affect the binding affinity of the complementary bispecific antibody to FcRn. In some embodiments, the introduction of L234F/L235E/P331S mutations in the heavy chain constant region of the anti-HER2 complementary bispecific antibody can significantly reduce the ADCC activity of the complementary bispecific antibody. In one embodiment, compared to an antibody whose Fc region does not contain the amino acid mutations, the ADCC activity of the complementary bispecific antibody is significantly weakened or undetectable, and the weakening or elimination of ADCC activity may be caused by the significantly reduced binding affinity of the complementary bispecific antibody to Fc ⁇ R.
  • the anti-HER2 complementary bispecific antibody comprises a light chain constant region, and the light chain constant region is selected from the Kappa constant region (as shown in SEQ ID NO: 233).
  • the anti-HER2 complementary bispecific antibody of the present invention comprises four polypeptide chains, wherein two polypeptide chains comprise VH1-L1-VH2-C-(Fc)n, VH1 represents the heavy chain variable region of the first antigen binding domain of D3 that specifically binds to the extracellular region of HER2, L1 represents a linker, VH2 represents the heavy chain variable region of the second antigen binding domain of D1 that specifically binds to the extracellular region of HER2, C represents the heavy chain constant region CH1, Fc represents the heavy chain constant region Fc domain, and n is 0 or 1; and the other two polypeptide chains comprise VL1-L2-VL2-CL, wherein VL1 represents the light chain variable region of the first antigen binding domain of D3 that specifically binds to the extracellular region of HER2, L2 represents a linker, VL2 represents the light chain variable region of the second antigen binding domain of D1 that specifically binds to the extracellular region of HER2, and CL is an I
  • the VH1 comprises the HCDR1 amino acid sequence as shown in SEQ ID NO:11, the HCDR2 amino acid sequence as shown in SEQ ID NO:24, 29, 30, or 31, and the HCDR3 amino acid sequence as shown in SEQ ID NO:37, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, and HCDR3, respectively;
  • L1 comprises the linker amino acid sequence as shown in SEQ ID NO:229;
  • VH2 comprises the linker amino acid sequence as shown in SEQ ID NO: NO:98, the HCDR1 amino acid sequence as shown in SEQ ID NO:102, the HCDR2 amino acid sequence as shown in SEQ ID NO:110, and the HCDR3 amino acid sequence as shown in SEQ ID NO:110, or amino acid sequences that are at least 85%, 86%, 8
  • the VH1 comprises the VH amino acid sequence as shown in SEQ ID NO:73, 78, 79, or 80, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto;
  • the VH2 comprises the VH amino acid sequence as shown in SEQ ID NO:169, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.
  • the VL1 comprises the LCDR1 amino acid sequence as shown in SEQ ID NO:42, the LCDR2 amino acid sequence as shown in SEQ ID NO:46 and the LCDR3 amino acid sequence as shown in SEQ ID NO:49, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the LCDR1, LCDR2 and LCDR3, respectively;
  • L2 comprises the linker amino acid sequence as shown in SEQ ID NO:230;
  • VL2 comprises The LCDR1 amino acid sequence as shown in SEQ ID NO:115, the LCDR2 amino acid sequence as shown in SEQ ID NO:133 and the LCDR3 amino acid sequence as shown in SEQ ID NO:142, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the VL1 comprises the VL amino acid sequence of SEQ ID NO:92, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto;
  • the VL2 comprises the VL amino acid sequence of SEQ ID NO:193, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Amino acid sequence.
  • the anti-HER2 complementary bispecific antibody of the present invention can specifically bind to two different epitopes of the HER2 extracellular region at the same time, so it can effectively cross-link the HER2 on the cell surface to form antigen-antibody cross-linked aggregates or clusters, thereby inducing cell endocytosis and promoting the internalized clusters to be transported to lysosomes and degraded in lysosomes.
  • the complementary bispecific antibody can significantly downregulate the expression of HER2 on the cell surface, reduce the dimerization of HER2, and thus inhibit the proliferation of tumor cells overexpressing HER2.
  • the anti-HER2 complementary bispecific antibody of the present invention does not interfere with the HER2-mediated signal transduction pathway, so it does not interfere with the normal biological function of HER2 in normal tissues/cells.
  • the present invention further provides an anti-HER2 complementary bispecific ADC, which comprises a small molecule toxin compound coupled to an anti-HER2 complementary bispecific antibody of the present invention via a cleavable linker, and has one or more of the following functional properties:
  • the anti-HER2 complementary bispecific ADC is a tetravalent molecule that can specifically bind to two non-overlapping epitopes of HER2 on the surface of tumor cells, and thus can effectively cross-link HER2 and form cross-linked aggregates or clusters on the tumor cell membrane.
  • the formation of clusters leads to rapid and efficient endocytosis by cells, and the intracellular transport pathway after endocytosis is changed from the usual recycling to lysosomal trafficking, which significantly improves the efficiency of the ADC transport into lysosomes, thereby releasing more small molecule toxin compounds into the cytoplasm after degradation in lysosomes.
  • the complementary bispecific ADC has stronger targeted cytotoxicity, which is manifested as stronger killing activity in HER2-overexpressing tumor cells (IHC 3 + or IHC 2 + /FISH + ) than existing HER2 targeted drugs (e.g., Trastuzumab, Pertuzumab, T-DM1, DS-8201), and can also play a direct killing role in HER2-low-expressing tumor cells (IHC 2 + /FISH - or IHC 1 + ), and has the potential to expand the scope of indications for HER2 targeted therapy; on the other hand, the released small molecule toxin compound is hydrophobic and has cell membrane permeability, so it can passively diffuse into the tumor microenvironment to exert a bystander effect (Bystander Effect), that is, killing adjacent tumor cells that express lower levels or do not express HER2.
  • the complementary bispecific ADC has a broader spectrum of tumor cell killing activity than ordinary ADCs, and is more difficult to produce drug resistance.
  • the anti-HER2 complementary bispecific ADC has a killing effect on tumors that have become resistant or recurrent to Trastuzumab, Pertuzumab, T-DM1 and DS-8201, and thus can solve the drug resistance problem of existing HER2 targeted drugs.
  • the anti-HER2 complementary bispecific ADC does not affect the dimerization of HER2 and the regulation of its mediated signal transduction pathways, and therefore does not interfere with the normal biological function of HER2 in cardiomyocytes, thereby greatly reducing the possibility of causing cardiac toxic side effects.
  • the anti-HER2 complementary bispecific ADC has significantly reduced Fc receptor binding affinity and thus has extremely low Fc receptor-mediated safety risks.
  • the ADC can be represented by formula (I):
  • Ab represents the anti-HER2 complementary bispecific antibody of the present invention
  • D represents a small molecule toxin compound (Drug);
  • L represents a cleavable linker that couples Ab to D
  • p represents the number of copies of (L-D) coupled to Ab, which ranges from 2 to 8.
  • Ab in the ADC is an anti-HER2 complementary bispecific antibody of the present invention, which comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 2, which can specifically bind to D3 of the extracellular region of HER2, and is an Fv domain; the second antigen-binding domain comprises VH and VL of the anti-HER2 antibody or its antigen-binding fragment shown in Table 3, which can specifically bind to D1 of the extracellular region of HER2, and is a Fab domain or an IgG domain.
  • the first antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise: (i) HCDR1 as shown in SEQ ID NO: 11; (ii) HCDR2 as shown in SEQ ID NO: 24, 29, 30, or 31; (iii) HCDR3 as shown in SEQ ID NO: 37; (iv) LCDR4 as shown in SEQ ID NO: 42.
  • DR1 (v) LCDR2 as shown in SEQ ID NO:46; and (vi) LCDR3 as shown in SEQ ID NO:49; or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to those of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.
  • the second antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise: (i) HCDR1 as shown in SEQ ID NO:98; (ii) HCDR2 as shown in SEQ ID NO:102; (iii) HCDR3 as shown in SEQ ID NO:110; (iv) LCDR1 as shown in SEQ ID NO:115; (v) v) LCDR2 as shown in SEQ ID NO:133; and (vi) LCDR3 as shown in SEQ ID NO:142; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.
  • the first antigen binding domain of the anti-HER2 complementary bispecific ADC comprises a VH as 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/or a VL as shown in SEQ ID NO: 92, 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 second antigen binding domain of the anti-HER2 complementary bispecific ADC comprises VH as 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; and/or VL as 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 thereto.
  • D in the ADC is a small molecule toxin compound, which has cytotoxic or cell growth inhibitory effects on both tumor cells and normal cells when it is not conjugated to the anti-HER2 complementary bispecific antibody of the present invention, but after the small molecule toxin compound is conjugated to form an ADC, it has cytotoxic and/or bystander killing effects only when the ADC is internalized and transported into the lysosome of the target cell expressing HER2 and dissociated from the ADC by enzymatic action, hydrolysis, oxidation or any other mechanism.
  • the small molecule toxin compound includes cytotoxins and chemotherapeutic drugs.
  • the cytotoxins include tubulin inhibitors and DNA damaging agents
  • the tubulin inhibitors include Eribulin, Auristatins derivatives (e.g., MMAE, MMAF, MMAD), Tubulysins, Cryptomycins and Maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4)
  • the DNA damaging agents include topoisomerase inhibitors (e.g., camptothecin derivatives SN-38, Exatecan and DXd), pyrrolobenzodiazepines (PBD), and Calicheamicin and its derivatives (e.g., N-acetyl Calicheamicin [CMC]), Duocarmycin, and the preferred small molecule toxin compound is Eribulin.
  • eribulin refers to a synthetic analog of halichondria B, a macrocyclic compound isolated from the marine sponge Halichondria okadais. As an inhibitor of microtubule dynamics, eribulin inhibits the formation of mitotic spindles by binding to tubulin, causing the cell division cycle to arrest at the G2/M phase.
  • the structures of exemplary eribulin or its analogs and methods for their synthesis are described in WO1999065894, WO2004034990, ZL201910197071.8, and/or ZL201910509222.9, the disclosures of which are incorporated herein by reference.
  • the small molecule toxin compound is eribulin, which has the characteristics of a large clinical therapeutic window and low off-target cytotoxicity.
  • the chemotherapeutic drug can be a natural or synthetic compound, including but not limited to alkyl chemotherapeutic agents and other compounds with alkylated forms (e.g., nitrogen mustards, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, dacarbazine), antimetabolites (e.g., folic acid, purine or pyrimidine antagonists), mitotic inhibitors (e.g., vinca alkaloids and derivatives of podophyllotoxin), cytotoxic antibiotics (e.g., anthracycline antibiotics including daunorubicin and doxorubicin, as well as actinomycin, bleomycin).
  • alkyl chemotherapeutic agents and other compounds with alkylated forms e.g., nitrogen mustards, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, dacarbazine
  • the cleavable linker is stable outside the tumor cells expressing HER2, so that the ADC of the present invention maintains structural stability in vitro or in the blood circulation, and will not cause systemic toxicity (off-target effect) due to the non-targeted or random release of small molecule toxin compounds. At the same time, after endocytosis into tumor cells expressing HER2, it can quickly enter the lysosome for degradation and release small molecule toxin compounds to kill tumor cells.
  • the cleavable linker refers to any linker comprising a cleavable portion.
  • cleavable portion refers to any cleavable chemical bond, for example, well known in the art, including but not limited to, bonds unstable to acid, bonds unstable to proteases/peptidases, bonds unstable to light, bonds unstable to esterases, and disulfide bonds.
  • the linker comprising a cleavable portion can allow small molecule toxin compounds to dissociate from the ADC via cleavage at specific sites in the linker.
  • the cleavable linker comprises a cleavable peptide portion, which can be cleaved by an intracellular protease (e.g., an endosomal protease, a lysosomal protease, or a tumor-associated protease), and then a small molecule toxin compound can be dissociated from the ADC to kill tumor cells.
  • the cleavable peptide portion comprises an amino acid unit that can be cleaved by a lysosomal cysteine cathepsin (e.g., cathepsin B, C, F, H, K, L, O, S, V, X, or W).
  • the amino acid unit may comprise naturally occurring amino acid residues and/or non-naturally occurring amino acid analogs, such as citrulline (Citrulline, referred to as Cit).
  • the amino acid unit comprises a dipeptide, a tripeptide, or a tetrapeptide, for example, a Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG) amino acid sequence that can be cleaved by cathepsin B, and preferred amino acid units include VC and GGFG.
  • the cleavable linker may comprise at least one spacer for coupling a small molecule toxin compound (D) to the anti-HER2 complementary bispecific antibody (Ab) of the present invention, wherein the spacer comprises a spacer conjugated to an antibody and/or a spacer conjugated to a small molecule.
  • the second spacer region to which the daughter compound is conjugated may comprise at least one spacer for coupling a small molecule toxin compound (D) to the anti-HER2 complementary bispecific antibody (Ab) of the present invention, wherein the spacer comprises a spacer conjugated to an antibody and/or a spacer conjugated to a small molecule. The second spacer region to which the daughter compound is conjugated.
  • the spacer conjugated to the antibody may be hydrophilic to increase the hydrophilicity of the ADC and improve its stability, reduce ADC product aggregation and reduce immunogenicity
  • an exemplary spacer comprises one or more polyethylene glycols (PEGs), such as 1, 2, 3, 4, 5, or 6 PEGs, preferably 2 PEGs.
  • PEGs polyethylene glycols
  • the spacer is attached to the antibody via maleimide (Mal), wherein the spacer attached to the antibody via Mal may also be referred to herein as a "Mal-spacer".
  • the Mal-spacer comprises one or more PEG moieties (e.g., 2 PEGs).
  • the second spacer conjugated to the small molecule compound is used to attach the cleavable portion of the cleavable linker (e.g., a cleavable peptide) to the small molecule compound.
  • the second spacer conjugated to the small molecule compound has the characteristics of self-immolation, which is conducive to the complete release of the small molecule compound in the target cell, that is, the small molecule compound released in the target cell does not carry the spacer portion or other modifying groups conjugated thereto, so that the released small molecule compound does not affect its anti-tumor activity due to the modified group.
  • the self-immolation spacer comprises a p-aminobenzyl unit, p-aminobenzyl alcohol (PABOH) is conjugated to the cleavable portion of the cleavable linker (e.g., the amino acid unit of the cleavable peptide) via an amide bond, and a carbamate group, a methylcarbamate group or a carbonate group is formed between PABOH and the small molecule compound (Hamann et al., Expert Opinion on Therapeutic Patents 2005, 15: 1087-1103).
  • PABOH p-aminobenzyl alcohol
  • the self-immolative fragmentation spacer comprises a p-aminobenzylcarbonyl (PAB) group represented by formula (III), and the self-immolative fragmentation of PAB involves a spontaneous 1,6-elimination reaction (Jain et al., Pharm Res 2015, 32: 3526-3540):
  • PAB p-aminobenzylcarbonyl
  • the cleavable linker comprises a Mal-spacer and a cleavable peptide portion.
  • the spacer comprises a PEG portion (e.g., 2 PEGs), and the cleavable peptide portion comprises an amino acid unit (e.g., a dipeptide VC and a tetrapeptide GGFG).
  • the cleavable linker comprises a covalently linked Mal-spacer-amino acid unit, wherein the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG.
  • the cleavable linker comprises a covalently linked Mal-spacer-amino acid unit-PAB, wherein the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG.
  • the spacer is (PEG) m , m is an integer of 1-5, preferably 2.
  • the cleavable linker comprises Mal-(PEG) 2 and VC.
  • the cleavable linker comprises Mal-(PEG) 2 and GGFG.
  • the cleavable linker comprises the structure: Mal-spacer-cleavable peptide moiety. In some embodiments, the cleavable linker comprises the structure: Mal-(PEG) 2 -VC and Mal-(PEG) 2 -GGFG.
  • the cleavable linker comprises a Mal-spacer, a cleavable peptide portion, and a second spacer.
  • the spacer comprises a PEG portion (e.g., 2 PEGs)
  • the cleavable peptide portion comprises an amino acid unit (e.g., a dipeptide VC)
  • the second spacer is self-immolative (e.g., PAB).
  • the cleavable linker comprises Mal-(PEG) 2 , VC, and PAB.
  • the cleavable linker comprises the structure: Mal-spacer-cleavable peptide moiety-second spacer. In some embodiments, the cleavable linker comprises the structure: Mal-(PEG) 2 -VC-PAB.
  • the Mal-spacer of the cleavable linker is coupled to one or more amino acid residues of the antibody portion in the ADC of the present invention, for example, the Mal-spacer can be coupled to the antibody via a thiol group, including coupling to the thiol group of one or more cysteines of the antibody.
  • the maleimide group of the Mal-spacer reacts with the thiol group of the cysteine residue of the antibody, thereby conjugating the Mal-spacer to the antibody.
  • the maleimide group of the Mal-spacer can react with the thiol group of the cysteine residue at a specific position in the constant region and/or variable region of the antibody, and further, the maleimide group of the Mal-spacer can be coupled to the free cysteine residue released after the disulfide bond and/or interchain disulfide bond of the hinge region of the antibody is reduced.
  • the maleimide group of the Mal-spacer is coupled to the cysteine residue in the hinge region of the antibody, and the interchain disulfide bonds in the hinge region of the antibody can be subjected to a chemical reaction (e.g., reduction, pH adjustment, or hydrolysis) to generate a plurality of free cysteine residues, or the amino acid residues at one or more specific sites of the antibody constant region can be subjected to DNA recombination technology (e.g., by substitution or insertion of cysteine residues) to generate engineered cysteine residues.
  • a chemical reaction e.g., reduction, pH adjustment, or hydrolysis
  • DNA recombination technology e.g., by substitution or insertion of cysteine residues
  • the second spacer of the cleavable linker is coupled to a small molecule compound, and the small molecule compound is eribulin or a derivative thereof.
  • the second spacer couples the cleavable peptide portion in the cleavable linker to the C-35 amine of eribulin, and the second spacer is self-immolative (e.g., PAB), and the cleavable peptide portion comprises VC.
  • the self-immolative spacer spontaneously undergoes self-immolative fragmentation after the cleavable peptide portion is enzymatically hydrolyzed, resulting in the release of eribulin from the ADC in a naturally active form.
  • the self-immolative spacer is PAB
  • the cleavable peptide portion VC is conjugated to the C-35 amine of eribulin to construct an ADC comprising a Mal-(PEG) 2 -VC-PAB-Eribulin structure.
  • the cleavable portion of the cleavable linker can be directly conjugated to the small molecule toxin compound portion of the ADC, wherein the small molecule toxin compound is eribulin or a derivative thereof.
  • the polypeptide includes an amino acid unit, wherein the amino acid unit includes GGFG, and the tetrapeptide is conjugated to the C-35 amine of Eribulin via a carboxyl group to construct an ADC including a Mal-(PEG) 2 -GGFG-Eribulin structure.
  • the GGFG tetrapeptide in the cleavable linker is directly conjugated to Eribulin, which can also ensure that the cleavable linker of the ADC can completely release Eribulin in a natural active form after cleavage in the target cell, so that it can play a role in targeted killing of tumor cells and/or a bystander effect.
  • the ADC structure of the present invention comprises the anti-HER2 complementary bispecific antibody of the present invention conjugated to Eribulin or its derivatives via a cleavable linker, wherein the cleavable linker comprises a Mal-spacer, a cleavable peptide portion and/or a second spacer.
  • the ADC structure comprises the anti-HER2 complementary bispecific antibody of the present invention conjugated to the C-35 amine of Eribulin via a cleavable linker, wherein the cleavable linker comprises Mal-(PEG) 2 -VC-PAB or Mal-(PEG) 2 -GGFG, and the free cysteine residue released after reduction of the disulfide bonds and/or interchain disulfide bonds in the hinge region of the antibody is conjugated to the Mal of the cleavable linker.
  • the ADC of the present invention comprises the following parts:
  • the ADC of the present invention comprises the anti-HER2 complementary bispecific antibody of the present invention coupled to a drug payload, wherein the drug payload comprises Mal-(PEG) 2 -VC-PAB-Eribulin and Mal-(PEG) 2 -GGFG-Eribulin.
  • the ADC comprises the anti-HER2 complementary bispecific antibody of the present invention conjugated to Mal of the drug payload via its cysteine residue, and further, the free cysteine residue released after reduction of the disulfide bond and/or interchain disulfide bond in the hinge region of the complementary bispecific antibody is conjugated to Mal of the drug payload.
  • the invention provides an ADC having the formula:
  • Ab represents the anti-HER2 complementary bispecific antibody described in the present invention, and p is 2 to 8.
  • the p in the formula (I) is also referred to herein as the drug-to-antibody ratio (DAR) or the number of payload molecules, that is, the number of small molecule compounds coupled to the antibody, and the p is 2 to 8, for example, 4 to 8.
  • DAR value of the anti-HER2 complementary bispecific antibody conjugated to eribulin via a cleavable linker contained in the ADC of the present invention has desirable characteristics, that is, the ADC has not only sufficient killing activity but also sufficient stability.
  • a higher DAR value may cause the ADC to aggregate and precipitate in an aqueous environment due to hydrophobic effects due to the strong hydrophobicity of the small molecule toxin compound, and may even cause safety risks in vivo;
  • a lower DAR value (for example, p ⁇ 2) may result in a lower killing activity of the ADC against tumor cells and affect the efficacy.
  • the optimal DAR value is about 4 to 8.
  • the average DAR value (ie, the average number of effective load molecules or average p) of the anti-HER2 complementary bispecific ADC of the present invention can be obtained by calculation using the results of conventional analytical methods known in the art (eg, reverse phase LC-MS mass spectrometry and/or HIC-HPLC).
  • the present invention provides a nucleic acid encoding an anti-HER2 antibody or an antigen-binding fragment thereof, or an anti-HER2 complementary bispecific antibody.
  • the present invention also includes polynucleotide variants encoding the amino acid sequences described herein.
  • the nucleotide sequence corresponding to the amino acid sequence described in the present invention, used as a probe or primer for nucleic acid isolation, or provided by the database for query can be obtained by back translation of the amino acid sequence.
  • the polymerase chain reaction (PCR) program can be used to separate and amplify the DNA sequence encoding the anti-HER2 antibody or its antigen-binding fragment, and the anti-HER2 complementary bispecific antibody of the present invention.
  • Oligonucleotides that define the desired ends of the DNA fragment combination are used as 5' and 3' primers.
  • the oligonucleotides may additionally contain a recognition site for a restriction endonuclease to facilitate the insertion of the amplified DNA fragment combination into an expression vector.
  • the nucleic acid molecules of the present invention include single-stranded and double-stranded forms of DNA and RNA, as well as corresponding complementary sequences, including isolated nucleic acid molecules, preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and whose quantity or concentration is capable of identifying, manipulating and recovering its component nucleotide sequences by standard biochemical methods (e.g., the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989 2nd edition, Cold Spring Harbor Laboratory).
  • sequences include open reading frames that are provided and/or constructed in the form of internal non-translated sequences or introns that are not commonly found in eukaryotic genes.
  • the sequence of non-translated DNA may be present at 5' or 3' of the open reading frame, wherein the sequence does not interfere with the manipulation or expression of the coding region.
  • the anti-HER2 antibody or its antigen-binding fragment of the present invention, or the anti-HER2 complementary bispecific antibody is prepared by the following steps: using PCR mutagenesis or other techniques known to those of ordinary skill in the art to carry out site-specific mutagenesis of nucleotides in the DNA encoding the anti-HER2 antibody or its antigen-binding fragment or the anti-HER2 complementary bispecific antibody to produce DNA encoding variants, and thereafter expressing the recombinant DNA in the cell culture as outlined herein.
  • the anti-HER2 antibody or its antigen-binding fragment and the anti-HER2 complementary bispecific antibody can also be prepared by in vitro synthesis using established techniques.
  • the anti-HER2 antibody or antigen-binding fragment thereof or anti-HER2 complementary bispecific antibody of the present invention is encoded by a very large number of nucleic acids, and the nucleic acids of each part are within the scope of the present invention and can be made using standard techniques. Therefore, having identified a specific amino acid sequence, those skilled in the art can simply modify one or more codons of their respective coding sequences in a manner that does not change the amino acid sequence of the anti-HER2 antibody or antigen-binding fragment thereof or anti-HER2 complementary bispecific antibody of the present invention to prepare many different nucleic acids.
  • the present invention also provides an expression vector of a nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody described herein.
  • the nucleic acid encoding the anti-HER2 antibody or its antigen-binding fragment of the present invention or the anti-HER2 complementary bispecific antibody can be constructed in a suitable vector to be introduced into a host cell for expression of the target protein.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the nucleic acid encoding the target protein in the vector is operably connected to the promoter.
  • operably linked refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription of the other nucleic acid sequence. recording and/or translation.
  • a nucleic acid expression control sequence e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites
  • Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • the vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes or protein shells.
  • the present invention also provides a host cell comprising a nucleic acid or an expression vector encoding the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody of the present invention.
  • the cell can be a eukaryotic cell, for example, a mammalian host cell, including but not limited to, SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J Gen Virol 1977, 36:59-74), baby hamster kidney cell (BHK-21, ATCC, CCL-10), Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), mouse testicular supporting cell (TM4, Mather, Biol Reprod 1980, 23:243-251).
  • SV40 transformed monkey kidney cell CV1 line COS-7, ATCC, CRL-1651
  • human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J Gen Virol 1977, 36:59-74
  • baby hamster kidney cell BHK-21, ATCC,
  • monkey kidney cells (CV1, ATCC, CCL-70), African green monkey kidney cells (VERO-76, ATCC, CRL-1587), human cervical cancer cells (HELA, ATCC, CCL-2), canine kidney cells (MDCK, ATCC, CCL-34), Buffalo rat liver cells (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human liver cancer cell line (HepG2, ATCC, HB-8065), mouse mammary tumor (MMT060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC5 cells, or FS4 cells.
  • CV1, ATCC, CCL-70 African green monkey kidney cells
  • HELA human cervical cancer cells
  • BBL 3A Buffalo rat liver cells
  • human lung cells W138, ATCC, CCL-75
  • human liver cancer cell line HepG2, ATCC, HB-8065
  • mouse mammary tumor
  • the anti-HER2 antibody provided by the present invention can be a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the method of producing monoclonal antibodies is 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 a monoclonal antibody that specifically binds to HER2.
  • a polypeptide of any fragment in the HER2 amino acid sequence (preferably the extracellular region of HER2) can be selected as the target antigen to immunize mice, and the antibody variable region genes can be amplified from the mature B cells of the mice, and the mouse antibody variable regions can be displayed on the surface of phages by phage display technology to construct a phage display library, and panning can be carried out by using specific target molecules (for example, the target antigen used to immunize mice), and the interaction between the antibody variable regions displayed on the surface of the phage and the target antigen is detected, and the antibody variable region library is screened and amplified by an in vitro selection method, and the in vitro selection is similar to natural selection.
  • specific target molecules for example, the target antigen used to immunize mice
  • the present invention constructs a phage display library and performs panning for the target antigen to screen out antibodies that recognize the target antigen from the antibody library.
  • the antibodies of the present invention can be produced using a variety of phage display methods known in the art.
  • phage display libraries can also be constructed according to the methods described in "Antibody Phage Display: Methods and Protocols (O'Brien and Aitken ed.)".
  • the nucleic acid encoding the variable region of the antibody can be inserted into the phage coat protein gene so that the phage displays the variable region of the antibody on its surface. At the same time, the nucleic acid encoding the variable region of the antibody is contained inside the phage, thereby realizing the connection between the phenotype and genotype of the variable region of the antibody.
  • phage display a large library of VH and/or VL regions of antibodies (or single-chain antibodies) can be expressed on the surface of filamentous phage particles, thereby pairing them to form binding domains. Phages can be screened from the library based on recognition and binding to the target antigen and the binding domains displayed.
  • the panning can be achieved by phage infection of host bacteria and reproduction and amplification in the host. After the phages secreted by the host bacteria displaying single-chain antibody fragments on their surfaces are collected, multiple rounds of panning can be performed as needed until phages that can selectively or specifically bind to the target antigen are obtained, and finally the amino acid sequence of the antibody variable region is obtained by sequencing the antibody gene in the phage genome (Arap et al., Science 1998, 279: 377-380; Smith et al., Science 1985, 228: 1315-1317).
  • the present invention uses HER2 extracellular region recombinant protein as the target antigen to immunize mice, obtains antibody heavy chain variable region family and light chain variable region family gene fragments from the spleen tissue of the immunized mice, and prepares scFv fragments based on this, and connects to the phage surface structural protein gene III, and uses co-expression to participate in phage assembly and display on the phage surface, thereby constructing a phage display library.
  • phage display library for a specific target antigen (for example, an antigen used to immunize mice), through multiple rounds of adsorption-elution-amplification repetitive processes (panning), phages that can specifically bind to the target antigen can be enriched, and then the corresponding DNA sequence information is obtained through gene sequencing technology, and then the amino acid sequence of the antibody variable region is inferred.
  • a specific target antigen for example, an antigen used to immunize mice
  • panning phages that can specifically bind to the target antigen can be enriched, and then the corresponding DNA sequence information is obtained through gene sequencing technology, and then the amino acid sequence of the antibody variable region is inferred.
  • the present invention provides a method for preparing the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody of the present invention using the host cell.
  • the method comprises transfecting a nucleic acid or expression vector encoding the anti-HER2 antibody or antigen-binding fragment thereof, or the anti-HER2 complementary bispecific antibody of the present invention into a host cell, and culturing the host cell in a culture medium for a period of time to express the anti-HER2 antibody or antigen-binding fragment thereof of the present invention.
  • the cell culture medium may be a commercially available culture medium without limitation.
  • the expressed anti-HER2 antibody or antigen-binding fragment thereof, or anti-HER2 complementary bispecific antibody can be secreted into the culture medium in which the host cells are grown.
  • the antibody is recovered from the culture medium using conventional protein purification methods, such as removing impurities by centrifugation or ultrafiltration, or purifying the resultant by affinity chromatography; other purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography, can also be used.
  • the ADC of the present invention can be prepared by any method known in the art, including but not limited to: (1) the nucleophilic group or electrophilic group of the antibody reacts with the cleavable linker to form an antibody-cleavable linker intermediate (Ab-L) via a covalent bond, and then reacts with a small molecule toxin compound (D).
  • the intermediate Ab-L may or may not be purified before reacting with the small molecule toxin compound; (2) the nucleophilic group or electrophilic group of the small molecule toxin compound reacts with the cleavable linker to form a small molecule toxin compound-cleavable linker intermediate (D-L) via a covalent bond, and then reacts with the nucleophilic group or electrophilic group of the antibody.
  • the intermediate D-L may or may not be purified before reacting with the antibody; or (3) the antibody, the cleavable linker and the small molecule toxin compound are mixed and reacted so that covalent bonds are formed between the antibody and the cleavable linker and between the cleavable linker and the small molecule toxin compound at the same time, thereby preparing the ADC of the present invention.
  • Several specific examples of methods for preparing ADC are known in the art, such as those described in US8624003 (one-pot method), US8163888 (one-step method) and US5208020 (two-step method).
  • the antibody is placed under reducing conditions before the conjugation reaction to produce one or more free cysteine residues.
  • a reducing agent e.g., dithiothreitol [DDT], 2-mercaptoethanol or tris (2-carboxyethyl) phosphine [TCEP]
  • DDT dithiothreitol
  • TCEP tris (2-carboxyethyl) phosphine
  • the reducing agent preferentially reduces the interchain disulfide bonds in the hinge region of the antibody, while the intrachain disulfide bonds of the antibody remain intact.
  • the reducing agent TCEP is used to react with the antibody to obtain a partially or completely reduced antibody with free sulfhydryl groups.
  • the chelating agent includes but is not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and the buffer includes but is not limited to histidine-hydrochloric acid, sodium phosphate, sodium borate, and sodium acetate solution.
  • the partially or completely reduced antibody with free thiol obtained above can react with a reactive functional group (e.g., maleimide group) of a cleavable linker (L) or a small molecule toxin compound-cleavable linker intermediate (D-L) to form a covalent bond (e.g., thioether bond) to obtain an antibody-cleavable linker intermediate (Ab-L) or ADC.
  • a reactive functional group e.g., maleimide group
  • a covalent bond e.g., thioether bond
  • the small molecule toxin compound-cleavable linker intermediate (D-L) is used interchangeably with the drug payload (Payload).
  • the purification method of the ADC prepared by the above method can be any biochemical method known in the art for purifying proteins or any combination thereof, including but not limited to affinity chromatography, ion exchange chromatography, mixed mode chromatography (e.g., ceramic hydroxyapatite chromatography), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation or any combination thereof.
  • affinity chromatography ion exchange chromatography
  • mixed mode chromatography e.g., ceramic hydroxyapatite chromatography
  • hydrophobic interaction chromatography e.g., size exclusion chromatography
  • dialysis e.g., dialysis, filtration, selective precipitation or any combination thereof.
  • the present invention provides a pharmaceutical composition, which comprises the anti-HER2 antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, or the anti-HER2 complementary bispecific ADC described in the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may contain any type of pharmaceutically acceptable carrier.
  • the carrier that can be used includes excipients, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents or combinations thereof.
  • suitable carriers Gennaro, Remington: The Science and Practice of Pharmacy, 2003 20th edition (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.
  • Parenteral administration refers to non-enteral and local modes of administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the antibody of the present invention can also be administered by non-parenteral routes (e.g., local, epidermal or mucosal administration routes), e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration.
  • non-parenteral routes e.g., local, epidermal or mucosal administration routes
  • intranasal, oral, vaginal, rectal, sublingual or topical administration e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration.
  • the active ingredient can be coated in the material to protect it from the effects of acid/base and other natural conditions that may inactivate it.
  • compositions can be sterile aqueous solutions or dispersions. They can also be in the form of microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentration.
  • the amount of active ingredient that can be combined with a carrier material to form a single dosage form is determined based on the subject and the specific mode of administration, and is generally the amount of the composition that can produce a therapeutic effect.
  • the composition formed with a pharmaceutically acceptable carrier contains 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% of the active ingredient.
  • the dosage regimen is adjusted to provide the best desired response (e.g., a therapeutic response). For example, a single dose may be administered, divided doses may be administered, or the dose may be proportionally reduced or increased depending on the treatment. It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and uniform dosage.
  • dosage unit form refers to physically discrete units that are suitable as unit doses for treating a subject; each unit dose contains a predetermined amount of active ingredient, which is calculated by the active ingredient and the desired amount.
  • the anti-HER2 antibody, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC of the present invention can also be administered as a sustained-release preparation, which can reduce the frequency of administration.
  • the dosage range of the anti-HER2 antibody or antigen-binding fragment thereof, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC of the present invention, or the composition comprising the anti-HER2 antibody or antigen-binding fragment thereof, anti-HER2 complementary bispecific antibody, or anti-HER2 complementary bispecific ADC can be about 0.0001 mg/kg to 100 mg/kg body weight, usually 0.001 mg/kg to 50 mg/kg body weight.
  • the "therapeutically effective dose” of the anti-HER2 antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, or the anti-HER2 complementary bispecific ADC of the present invention preferably can reduce the severity of disease symptoms, increase the frequency and duration of the disease symptom-free period, or prevent body damage or disability caused by disease distress.
  • the "therapeutically effective dose” of tumor-bearing subjects 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%.
  • the therapeutically effective amount of a therapeutic antibody or ADC can reduce the size of the tumor or improve the symptoms of the subject, who is usually a human or other mammal.
  • the "therapeutically effective dose” can also be determined differently according to various factors, including but not limited to formulation method, administration method, age, body, weight, patient's gender or pathological condition, diet, administration time, administration interval, administration route, excretion rate and reaction sensitivity.
  • the pharmaceutical composition may be selected as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. See, for example, Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, 1978 (Marcel Dekker).
  • the therapeutic drug composition can be delivered by the following medical devices, wherein the medical device is selected from: (1) needle-free subcutaneous injection devices (e.g., US5399163, US5383851, US5312335, US5064413, US4941880, US4790824 and US4596556); (2) microinfusion pumps (US4487603); (3) transdermal devices (US4486194); (4) infusion devices (US4447233 and US4447224); and (5) osmotic devices (US4439196 and US4475196); the disclosures of which are incorporated herein by reference.
  • needle-free subcutaneous injection devices e.g., US5399163, US5383851, US5312335, US5064413, US4941880, US4790824 and US4596556
  • microinfusion pumps US4487603
  • transdermal devices US4486194
  • infusion devices US4447233 and US4447224
  • osmotic devices US4439196 and US4475196
  • the anti-HER2 antibodies or antigen-binding fragments thereof, anti-HER2 complementary bispecific antibodies, or anti-HER2 complementary bispecific ADCs of the present invention can be formulated to ensure biodistribution in vivo.
  • the therapeutic antibodies or ADCs of the present invention cross the blood-brain barrier, they can be formulated into liposomes, which can additionally contain targeting moieties to enhance selective delivery to specific cells or organs.
  • the present invention provides a drug kit, which comprises an effective amount of the anti-HER2 antibody or antigen-binding fragment thereof, the anti-HER2 complementary bispecific antibody, the anti-HER2 complementary bispecific ADC, or the pharmaceutical composition of the present invention, and optionally at least one additional tumor therapeutic agent (i.e., the drug kit may contain or not contain at least one additional tumor therapeutic agent).
  • the tumor therapeutic agent may include but is not limited to another antagonist of ErbB2/HER2; EGFR antagonist, HER3 antagonist; MET antagonist, small molecule inhibitor of MET (e.g., Capmatinib); IGF1R antagonist (e.g., anti-IGF1R antibody); B-Raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720); PDGFR- ⁇ inhibitor (e.g., anti-PDGFR- ⁇ antibody); PDGFR- ⁇ inhibitor (e.g., anti-PDGFR- ⁇ antibody or small molecule agonist); enzyme inhibitors [e.g., imatinib mesylate or sunitinib malate]); PDGF ligand inhibitors (e.g., anti-PDGF-A antibody, anti-PDGF-B antibody, anti-PDGF-C antibody or anti-PDGF-D antibody, aptamer, siRNA, etc.); VEGF antagonists (e
  • the other ErbB2/HER2 antagonist includes an anti-HER2 monoclonal antibody (e.g., Trastuzumab, Pertuzumab) and/or ADC (e.g., T-DM1, DS-8201), or an anti-ErbB2/HER2 small molecule inhibitor (e.g., tyrosine kinase inhibitors such as lapatinib, pyrotinib, and neratinib);
  • the EGFR antagonist includes an anti-EGFR antibody (e.g., Cetuximab, Panitumumab), an anti-EGFR small molecule inhibitor (e.g., Gefitinib, Erlotinib), an anti-EGFRvIII antagonist (e.g., anti-EGFRvIII antibody);
  • the HER3 antagonist includes but is not limited to an anti-HER3 antibody (e.g., Patritumab);
  • the MET antagonist includes but is not limited to an anti-HER3 antibody (e.g.
  • the present invention relates to a method for treating a cancer expressing HER2, the method comprising administering an effective amount of the anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, or inventive pharmaceutical composition or kit of the present invention to a subject in need thereof.
  • the present invention relates to the use of the above-mentioned antibody, ADC, pharmaceutical composition or kit in the preparation of a drug for treating a cancer expressing HER2.
  • the present application relates to the above-mentioned antibody, ADC, pharmaceutical composition or kit for treating a cancer expressing HER2.
  • the cancer includes, but is not limited to, breast cancer, ovarian cancer, cervical cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, melanoma, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, endometrial cancer, and cancer at all stages, such as early cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission.
  • the cancer may be HER2 overexpressed or underexpressed.
  • the subject may be a human, non-human primate, or other mammal such as dog, mouse, or rat.
  • cancers expressing HER2 can be characterized by the amount of HER2 expression on the surface of cancer cells (i.e., by "HER2 status").
  • the expression of HER2 can be assessed by methods such as immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), etc.
  • the cancer includes cancers with overexpression of HER2 (e.g., IHC 3+ , or IHC 2 + /FISH + ), and/or cancers with low expression of HER2 (IHC 2 + / FISH- , or IHC 1+ ).
  • the anti-HER2 complementary bispecific ADC or its pharmaceutical composition has a killing effect on tumors/cancers that overexpress HER2 and/or tumors/cancers that underexpress HER2.
  • the anti-HER2 complementary bispecific antibody or pharmaceutical composition thereof has inhibitory activity on the growth or proliferation of tumor cells overexpressing HER2.
  • administering an effective amount of an anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, pharmaceutical composition, or kit of the invention to a subject in need thereof can reduce the subject's tumor volume, inhibit tumor growth, prolong the subject's disease-free survival or progression-free survival, increase the subject's overall survival, reduce tumor metastasis, or improve the subject's quality of life.
  • the present invention also relates to a method for treating cancer that is resistant to or relapses from existing HER2 targeted therapeutic agents, the method comprising administering an effective amount of the anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, pharmaceutical composition, or kit of the present invention to a patient in need thereof.
  • the present invention relates to the use of the antibody, ADC, pharmaceutical composition, or kit in the preparation of a drug for treating cancer that is resistant to or relapses from HER2 targeted therapeutic agents.
  • the patient does not respond or responds poorly to one or more existing HER2 targeted therapeutic agents, including Trastuzumab, Pertuzumab, T-DM1, DS-8201.
  • the non-response is manifested as an increase in the patient's tumor growth, tumor volume, tumor metastasis formation or number of metastases, and the non-response can also be a shortening of tumor metastasis development or disease progression time.
  • the poor response refers to the patient's tumor growth or metastasis in a short period of time during or after the standard therapy of the HER2 targeted therapeutic agent.
  • the present invention also relates to a method for treating patients who are not suitable for existing HER2 targeted therapy or are difficult to treat, or who develop drug resistance or relapse after receiving existing HER2 targeted therapy, the method comprising administering an effective amount of the anti-HER2 complementary bispecific antibody, anti-HER2 complementary bispecific ADC, pharmaceutical composition, or kit of the present invention to patients in need.
  • the present invention relates to the use of the antibody, ADC, pharmaceutical composition, or kit in the preparation of a drug for treating patients who are not suitable for existing HER2 targeted therapy or are difficult to treat, or who develop drug resistance or relapse after receiving existing HER2 targeted therapy.
  • the HER2 targeted therapy includes treatment with Trastuzumab, Pertuzumab, T-DM1, or DS-8201.
  • the anti-HER2 complementary bispecific antibodies, anti-HER2 complementary bispecific ADCs, pharmaceutical compositions, or kits described herein can be used to treat progressive cancer.
  • anti-HER2 complementary bispecific antibodies, anti-HER2 complementary bispecific ADCs, pharmaceutical compositions, or kits of the present invention can be used alone or in combination with other types of cancer therapies known in the art, such as surgery, chemotherapy, radiotherapy, gene therapy, immunotherapy, photodynamic therapy, radiofrequency ablation, etc.
  • the present invention also relates to the use of the anti-HER2 antibody or its antigen-binding fragment or the anti-HER2 complementary bispecific antibody for detecting and/or measuring HER2 or tumor cells expressing HER2 in a sample, and for screening cancer patients who are responsive to the anti-HER2 complementary bispecific ADC treatment of the present invention.
  • the present application relates to a method for detecting and/or measuring HER2 or tumor cells expressing HER2 in a sample, and a method for screening cancer patients who are responsive to the above-mentioned ADC treatment, comprising incubating the anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 complementary bispecific antibody with the sample or a biological sample separated from the patient, and detecting whether the antibody is bound to the sample or biological sample.
  • the present application relates to the above-mentioned anti-HER2 antibody or its antigen-binding fragment, or the anti-HER2 complementary bispecific antibody for detecting and/or measuring HER2 or tumor cells expressing HER2 in a sample, or screening cancer patients who are responsive to the above-mentioned ADC treatment.
  • the anti-HER2 antibody or antigen-binding fragment thereof, or anti-HER2 complementary bispecific antibody can be used to diagnose a disorder or disease of abnormal HER2 expression (e.g., overexpression, underexpression, or lack of expression) to facilitate determination of a treatment regimen.
  • the antibody can be conjugated with a detectable marker or reporter molecule, and the labeled antibody is contacted with a sample obtained from a patient to diagnose and determine HER2 expression.
  • the detectable marker or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I; a fluorescent material, such as umbelliferone, luciferin, rhodamine, fluorescein isothiocyanate, dichlorotriazinamine fluorescein, dansyl chloride or phycoerythrin; a chemiluminescent material, such as luminol; a bioluminescent material, such as luciferase, luciferin or aequorin; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase, horseradish peroxidase or luciferase.
  • a radioisotope such as 3 H, 14 C, 32 P, 35 S, or 125 I
  • a fluorescent material such as umbelliferone, luciferin, rhodamine, fluorescein
  • HER2 assays that can be used to detect or measure HER2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoPET (e.g., 89 Zr, 64 Cu, etc.) and fluorescence activated cell flow cytometry (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoPET immunoPET
  • FACS fluorescence activated cell flow cytometry
  • mice Two BALB/c mice were immunized with human HER2 extracellular region recombinant protein (sequence derived from position 23-652 of NP_004439.2). After three immunizations, blood was collected and the immune titer was tested by ELISA. The results showed that the immune titer was above 1:1,000,000 (data not shown). The mice were boosted with immunization, and the spleens of the mice were removed under sterile conditions 3 days later, and the single cell suspension of the spleens of the mice was prepared.
  • a reverse transcription kit Primerative TM II First Strand cDNA Synthesis Kit,
  • the two phage display libraries described in Item 1.1 of this Example were respectively selected using the HER2 extracellular domain recombinant protein as the target antigen. After 3 to 5 rounds of "adsorption-elution-amplification” selection, monoclonal ELISA screening and PCR enzyme digestion experiment verification, and sequencing analysis of the candidate clones, 15 sequence-specific anti-HER2 mouse monoclonal antibodies were finally obtained.
  • the light and heavy chain genes of the 15 mouse antibodies obtained by the above screening were homologously recombined with the linearized expression vector pcDNA3.1 (the heavy chain variable region [VH] was connected to the pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region [VL] was connected to the pcDNA3.1 vector containing the human CK constant region), and the chimeric antibody expression vector with the correct sequence was obtained by colony PCR detection and DNA sequencing analysis.
  • the chimeric antibody expression vector was extracted and purified using conventional methods, and then the 15 groups of chimeric antibody expression vectors containing light and heavy chain gene fragments were transiently transferred into ExpiCHO-S cells (Thermo), and the chimeric antibodies were expressed by serum-free culture.
  • the anti-HER2 chimeric antibody protein was obtained by Protein A affinity chromatography purification using the AKTA Pure system.
  • the binding activity of anti-HER2 chimeric antibodies to human ErbB/HER family members was detected by ELISA.
  • the specific method is as follows: dilute the HER2 extracellular domain recombinant protein and the extracellular domain recombinant proteins of other members of the human ErbB/HER family (EGFR, HER3 and HER4) with PBS, and then add 100 ⁇ L/well to a 96-well ELISA plate and incubate overnight at 4°C. After washing the plate with PBS, add 200 ⁇ L of blocking solution (PBST containing 1% BSA [PBS+0.1% Tween-20]) to each well and incubate at room temperature for 1 hour.
  • PBST containing 1% BSA [PBS+0.1% Tween-20]
  • test antibodies including anti-HER2 chimeric antibodies and control antibodies Trastuzumab, Cetuximab or Patritumab
  • test antibodies including anti-HER2 chimeric antibodies and control antibodies Trastuzumab, Cetuximab or Patritumab
  • 100 ⁇ L of anti-human IgG (H+L)-HRP antibody Jackson Immuno
  • NCI-N87 cells were collected, washed once with pre-cooled FACS buffer (PBS + 1% BSA), and then the cells were resuspended to 6-10 ⁇ 10 6 cells/mL, and added to a 96-well U-shaped plate at 50 ⁇ L/well, followed by adding 50 ⁇ L/well of gradient dilutions of the chimeric antibody to be tested or the positive control antibody Trastuzumab (with FACS buffer as the negative blank control), and incubated on ice for 60 minutes.
  • FACS buffer PBS + 1% BSA
  • the gene sequences of different subdomains of human and mouse HER2 were recombined and spliced, and homologous recombination was connected to the pcDNA3.1 vector to construct a human-mouse chimeric HER2 extracellular region recombinant protein expression plasmid ( Figure 3A), and transient expression was obtained to obtain the human-mouse chimeric HER2 extracellular region recombinant protein supernatant (the C-terminus of each recombinant protein was labeled with a 6 ⁇ His tag).
  • the human-mouse chimeric recombinant protein was used as an antigen to detect the binding epitope of the chimeric antibody in the HER2 extracellular region by ELISA.
  • the specific method is as follows: After the chimeric antibodies to be tested were diluted with PBS, they were added to a 96-well ELISA plate at 100 ⁇ L/well and incubated overnight at 4°C. After washing the plate with PBS, 200 ⁇ L of blocking solution (PBST containing 1% BSA) was added to each well and incubated at room temperature for 1 hour.
  • PBST containing 1% BSA
  • the recognition epitope of mAb2117 is located at D3 of the HER2 extracellular region
  • the recognition epitopes of mAb2126, mAb2164 and mAb2170 are located at D1 of the HER2 extracellular region
  • the recognition epitope of mAb2128 is located at D4 of the HER2 extracellular region.
  • the antigen binding competition activity between each chimeric antibody was detected by competitive ELISA.
  • the specific method is as follows: dilute the HER2 extracellular region recombinant protein with PBS, add 100 ⁇ L/well to a 96-well ELISA plate, and incubate overnight at 4°C. After washing the plate with PBS, add 200 ⁇ L of blocking solution (PBST containing 1% BSA) to each well and incubate at room temperature for 1 hour.
  • each chimeric antibody to be tested or the control antibody Trastuzumab diluted in the blocking solution at 50 ⁇ L/well (starting concentration is 50 ⁇ g/mL, 3-fold gradient dilution, a total of 7 concentration gradients), incubate at room temperature for 1 hour, add 50 ⁇ L of each biotin-labeled chimeric antibody to each well (final concentration is 0.5 ⁇ g/mL), and continue to incubate at room temperature for 1 hour.
  • 100 ⁇ L of HRP-Streptavidin (Jackson Immuno) diluted 1:10,000 in blocking solution was added to each well and incubated at room temperature for 1 hour.
  • TMB colorimetric solution was added to each well, incubated at room temperature in the dark for 3-10 minutes, and then 50 ⁇ L of stop solution (2M HCl) was added to each well, and the OD450 value was read using a multifunctional microplate reader (Varioskan, Thermo).
  • the binding kinetics of each chimeric antibody and the HER2 extracellular domain recombinant protein were detected using a molecular interaction instrument (ForteBio, model R8).
  • the specific method is as follows: the chimeric antibodies mAb2117, mAb2126, mAb2128, mAb2164, and mAb2170 to be tested were diluted to 5 ⁇ g/mL with PBS, and added to 96-well black-walled plates at 200 ⁇ L per well; then the HER2 extracellular domain recombinant protein was diluted to 200 nM with PBS, and added to 96-well black-walled plates at 200 ⁇ L per well after a 1:2 gradient dilution.
  • the 96-well black-walled plate containing the sample to be tested and the Protein A detection probe were placed in the detector, and the program was set to allow the Protein A probe to capture the antibody, followed by binding and dissociation detection with the antigen; after each binding and dissociation cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe, and then the next cycle of detection was started. After the test is completed, the analysis software is used to fit the binding constant and dissociation constant between the corresponding antibody and the antigen, and the affinity constant (K D ) value is calculated. The results are shown in Table 4.
  • the binding affinity constant (K D ) values of chimeric antibodies mAb2117, mAb2126, mAb2128, mAb2164, and mAb2170 to the HER2 extracellular region recombinant protein are all about 10 -9 M.
  • trastuzumab can induce phosphorylation of the Y1248 site of the HER2 intracellular domain in cardiomyocytes. This triggers a series of signal transduction cascade reactions, which ultimately lead to the destruction of the homeostasis of myocardial cells (Mohan et al., Molecular Cancer Therapeutics 2016, 15: 1321-1331). This mechanism may be one of the reasons why Trastuzumab causes cardiotoxicity. Therefore, this example evaluates whether anti-HER2 antibodies can induce phosphorylation of the HER2-Y1248 site of SKBR-3 cells to determine whether they have cardiotoxicity similar to that of Trastuzumab.
  • SKBR-3 cells are collected and resuspended in RPMI-1640 serum-free medium (containing 0.1% BSA), then inoculated into 6-well plates, and cultured overnight at 37°C.
  • the test antibody or control antibody (Trastuzumab, Pertuzumab) was added to each well at a final concentration of 4 ⁇ g/mL and incubated at 37°C for 30 minutes. Collect cells from each well in a 1.5-mL EP tube, wash twice with pre-cooled PBS, and add 150 ⁇ L RIPA cell lysis buffer (Porteintech) for total protein extraction.
  • CST rabbit anti-human HER2
  • Abcam rabbit anti-human HER2 antibody
  • NRG-1 can induce heterodimerization of HER2 and activate downstream signal transduction pathways, among which AKT phosphorylation is a landmark event in its downstream signal transduction cascade.
  • This example evaluates the effect of antibodies on HER2 receptor signal transduction pathways by detecting their inhibitory effects on NRG-1-induced AKT phosphorylation.
  • the specific method is as follows: T47D cells are inoculated into 6-well plates. After the cells grow to about 80% confluence, the culture medium is discarded and serum-free RPMI-1640 is added for starvation culture overnight. The test antibody or control antibody (Trastuzumab, Pertuzumab) is added to each well at a final concentration of 100nM.
  • NRG-1 NRG-1 (NOVUS) is added to each well at a final concentration of 100ng/mL and incubated for another 10 minutes. After washing the cells twice with pre-cooled PBS, whole protein extraction and Western-blot detection were performed according to the method described in Item 1.9 of this Example.
  • the primary antibody used was rabbit anti-p-AKT (Ser465) antibody (CST) or rabbit anti-pan-AKT antibody (CST), and the secondary antibody was goat anti-rabbit IgG-HRP antibody (Jacskon Immuno). The results are shown in Figure 6.
  • the chimeric antibodies mAb2117 and mAb2126 had no inhibitory effect on NRG-1-induced AKT phosphorylation, indicating that they do not affect NRG-1-induced HER2 heterodimerization and the activation of its downstream signal transduction pathways.
  • the heavy chain framework sequence of the chimeric antibody mAb2117 was replaced with the framework sequence of Germline IGHV3-23 by CDR transplantation, and was labeled Hu2117-H1; the light chain framework sequence of mAb2117 was replaced with the framework sequence of Germline IGKV1-39, and was labeled Hu2117-K1.
  • the mAb2117 chimeric antibody and its humanized antibody were respectively constructed into single-chain antibody fusion proteins of hIgG1Fc (scFv-hIgG1Fc fusion proteins), and the scFv antibody gene fragments were obtained by gene sequence synthesis, namely humanized single-chain antibodies Hu2117HK and Hu2117KH and chimeric single-chain antibody controls Mu2117HK and Mu2117KH, wherein HK represents the structure of the single-chain antibody as "(N-terminal) heavy chain variable region nucleic acid sequence-linker (G 4 S) 3 -light chain variable region nucleic acid sequence (C-terminal)", and KH represents the structure of the single-chain antibody as "(N-terminal) light chain variable region nucleic acid sequence-linker (G 4 S) 3 -heavy chain variable region nucleic acid sequence (C-terminal)".
  • HK represents the structure of the single-chain antibody as "(N-terminal) heavy chain variable region nucleic acid sequence-
  • the scFv gene fragments were homologously recombined in the pcDNA3.1 (containing human IgG1Fc sequence) vector to construct antibody expression plasmids. After the expression plasmids were extracted, they were transiently transferred into ExpiCHO-S cells (Thermo) for serum-free culture. The antibodies in the culture supernatant were purified by Protein A affinity chromatography using the AKTA Pure system to obtain single-chain antibody fusion proteins. The binding kinetics of the single-chain antibody fusion protein and the HER2 recombinant protein were analyzed using ForteBio. The test results are shown in Table 5. The humanized antibody basically retains the binding activity with HER2, and the affinity constant ( KD ) is about 10-7M .
  • the amino acid residues with low humanization degree, potential stability risk sites, and mouse amino acid residues in the framework region of the light and heavy chain variable region sequences of the humanized antibody Hu2117HK were analyzed, and a total of 50 single-point/multi-point mutations were designed to improve the humanization degree, antigen binding affinity and/or stability of the antibody.
  • the 50 designed single-point/multi-point mutations were introduced into the single-chain antibody Hu2117HK sequence by PCR, and the single-chain antibody mutant proteins were expressed.
  • the ForteBio molecular interaction instrument was used to detect the transient expression levels of the above 50 single-chain antibody mutants and their parent single-chain antibody Hu2117HK.
  • the specific method is as follows: after transient expression in ExpiCHO-S cells, the culture supernatant was harvested and added to a 96-well black board at 200 ⁇ L/well, the sample to be tested and the Protein A probe were placed in the ForteBio instrument, and the binding rate of each antibody molecule in the culture supernatant with the Protein A probe was detected using a preset quantitative program; after each cycle, the probe was immersed in 10mM glycine buffer (pH 1.5) to regenerate the probe, and then the next cycle detection was started.
  • the analysis software is used to calculate the expression level of each single-chain antibody according to the standard curve.
  • ForteBio was used to analyze the binding kinetics of each single-chain antibody mutant with HER2.
  • These antibodies include Hu2117HK-Mu01, -Mu02, -Mu03, -Mu04, -Mu05, -Mu06, -Mu08, -Mu09, -Mu10, -Mu11, -Mu12, -Mu13, -Mu17, -Mu20, -Mu21, -Mu23, -Mu24, -Mu25, -Mu27, -Mu28, -Mu32, -Mu33, -Mu36, -Mu37, -Mu38, -Mu39, -Mu40, -Mu45, -Mu46, -Mu47, -Mu48, -Mu49, and -Mu50 ( Figure 7A).
  • the above-mentioned preferred single-point/multi-point mutations were combined and introduced into the variable region sequence of the humanized single-chain antibody Hu2117HK to construct antibody molecules Hu2117-HK201 ⁇ -HK203 (abbreviated as HK201 ⁇ HK203) containing different combinations of mutations, and Hu2117-HK301 ⁇ -HK312 (abbreviated as HK301 ⁇ HK312).
  • the single-chain antibody samples were prepared according to the method described in item 2.2 of this embodiment, and ForteBio was used to detect the transient expression of each mutant and the parent single-chain antibody Hu2117HK.
  • the combined mutations and transient expression levels contained in each antibody molecule are shown in Table 7.
  • the antibodies were graded diluted according to the method described in Item 1.5 of Example 1, and the binding to BT474 cells and RT-112 cells was detected by flow cytometry, wherein the control antibody was the parent single-chain antibody Hu2117HK.
  • the results are shown in Table 8 and Figure 8, and these optimized humanized antibodies can bind to BT474 cells and RT-112 cells, and the binding activity is equivalent to that of the parent antibody Hu2117HK.
  • the heavy chain framework region sequence of the chimeric antibody mAb2126 was replaced by the framework region sequence of Germline IGHV4-4 or IGHV7-4 using the CDR transplantation method, and they were marked as Hu2126-H1 and Hu2126-H2 respectively; the light chain framework region sequence of mAb2126 was replaced by the framework region sequence of Germline IGKV1-39, and was marked as Hu2126-K1.
  • Humanized antibody variable region gene fragments were obtained by gene sequence synthesis, and the light and heavy chain gene fragments were respectively connected to the linearized expression vector pcDNA3.1 to construct antibody expression plasmids (the heavy chain variable region [VH] was connected to the pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region [VL] was connected to the pcDNA3.1 vector containing the human CK constant region).
  • the expression plasmids were extracted according to conventional methods and transiently transferred into ExpiCHO-S cells (Thermo) for serum-free expression culture. The culture supernatant was purified by Protein A affinity chromatography using the AKTA Pure system to obtain humanized antibodies Hu2126-H1K1 and Hu2126-H2K1.
  • Full R 2 indicates the similarity between the fitted curve and the measured curve.
  • the humanized antibody Hu2126-H2K1 was analyzed using the online software abYsis (http://abysis.org/abysis/index.html) and MOE software. After analyzing the amino acid residues with relatively low humanization degree in the light and heavy chain variable region sequences, potential molecular stability risk sites, and mouse amino acid residues in the framework region, a total of 63 single point mutations were designed to improve the degree of humanization, antigen binding affinity, and/or stability of the antibody. Specifically, 63 designed single point mutations were introduced into the humanized antibody Hu2126-H2K1 sequence by PCR, and expression plasmids were constructed.
  • the antibody light and heavy chain expression plasmids were combined and transiently transfected into ExpiCHO-S cells for antibody expression, and 63 antibody mutant expression supernatants containing single point mutations were obtained.
  • Item 2.2, ForteBio was used to detect the expression level of each antibody mutant after 3 days of transient culture and the binding kinetics with HER2.
  • the single point mutations in the light chain are combined and introduced into Hu2126-K1, and preferably, the single point mutations in the heavy chain are combined and introduced into Hu2126-H2, and the light chain mutation combination expression plasmid and the heavy chain mutation combination expression plasmid are constructed respectively, and then the light and heavy chain expression plasmids are paired and transiently transfected into ExpiCHO-S cells, and Hu2126-H2K1-L71-H72b-Mu10 ⁇ -Mu19 (referred to as 7172b-Mu10 ⁇ -Mu19) are obtained after culture expression and purification, a total of 10 antibody mutants.
  • the binding kinetics of the humanized antibody containing the combined mutations to HER2 were detected using ForteBio.
  • a pair of monospecific antibodies (Hu2117-HK304 and Hu2126-H2K1-L71-H72b-Mu14) that can respectively recognize different epitopes in the extracellular region of HER2 and do not compete with each other were selected to construct an exemplary complementary bispecific antibody.
  • the bispecific antibody configuration is a DVD-IgG structure
  • the Fv domain is the variable region sequence of Hu2117-HK304
  • the IgG domain is the Hu2126-H2K1-L71-H72b-Mu14 sequence
  • the heavy chain variable region of the Fv domain is connected to the heavy chain of the IgG domain through a linker ( G4S ) 1
  • the light chain variable region of the Fv domain is connected to the light chain of the IgG domain through a linker ( G4S ) 3.
  • L234F/L235E/P331S L234F/L235E/P331S (EU Numbering) mutations (referred to as "TM mutations") are introduced into the Fc region of the bispecific antibody molecule to reduce its binding to Fc ⁇ Rs and C1q, thereby eliminating the Fc effector function.
  • the constructed complementary bispecific antibody is named 04BS-109-WT.
  • the 04BS-109-WT bispecific antibody expression plasmid was transiently transfected into ExpiCHO-S cells for antibody protein expression and purified by Protein A affinity chromatography.
  • the 04BS-109-WT bispecific antibody molecule underwent colloidal precipitation during elution (the eluent did not contain NaCl), and the eluted product was turbid in the solution. However, after adding 0.1 M NaCl, the colloidal precipitate gradually dissolved, and the antibody solution finally became clear.
  • the anti-HER2 monospecific antibody derived from the Fv domain sequence of 04BS-1123-ST06 was named Hu2117-HK304-06.
  • Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 retain the property of their parent antibodies (chimeric antibodies mAb2117 and mAb2126) that do not compete for binding to HER2, this experiment was conducted according to the method described in Item 1.5 of Example 1.
  • the two optimized antibodies diluted from 800 ⁇ g/mL were mixed with 8 ⁇ g/mL biotin-labeled Hu2117-HK304-06 antibody in equal volumes and incubated with BT474 cells, and then AF488-labeled streptavidin (Jackson Immuno) was used as the secondary antibody to detect the competitive inhibition activity of the two antibodies on HER2-expressing tumor cells by flow cytometry.
  • AF488-labeled streptavidin Jackson Immuno
  • This example uses flow cytometry to measure the endocytosis rate of the above exemplary anti-HER2 complementary bispecific antibodies in cells expressing different levels of HER2 (including BT474, JIMT-1, and RT-112).
  • the specific method is as follows: After the cells are collected by trypsin digestion, they are washed with pre-cooled FACS buffer and resuspended to 6 to 10 ⁇ 10 6 cells/mL; the cell suspension is transferred to a 96-well U-shaped deep well plate at 300 ⁇ L/well, and then 300 ⁇ L of the antibody to be tested is added to each well (the final concentration of the antibody is 20 ⁇ g/mL), mixed and incubated on ice for 30 to 60 minutes; after washing twice with pre-cooled FACS buffer, the cells in each well are fully resuspended with 600 ⁇ L/well pre-cooled FACS buffer, and then the cell suspension is evenly distributed into 5 96-well U-shaped plates at 100 ⁇ L/well, and one of the plates is randomly selected as the
  • the internalization rate (percentage value) of the antibody was calculated by the following formula: (MFI control group -MFI experimental group )/MFI control group ⁇ 100%. The results are shown in Figure 11.
  • the anti-HER2 complementary bispecific antibodies 04BS-1123-ST04, 04BS-1123-ST05, 04BS-1123-ST06 and their parent antibody 04BS-109-WT can induce faster and more efficient endocytosis in BT474, JIMT-1 and RT-112 cells, among which the endocytosis rate of the complementary bispecific antibodies in JIMT-1 and RT-112 cells can reach about 60%, and the endocytosis rate in BT474 cells can be as high as about 80%.
  • the subcellular localization of the antibody was detected by confocal microscopy in this example.
  • the specific method is as follows: SKBR-3 cells are collected and plated into 5 8-well chamber slides (Thermo) for culture; after the cells grow into a uniform monolayer, the culture medium is discarded and rinsed once with PBS; 500 ⁇ L of 10 ⁇ g/mL anti-HER2 complementary bispecific antibody 04BS-1123-ST06, control antibody Trastuzumab, or human IgG isotype control antibody (such as WO2003106496A1) is added to the chamber of the chamber slide.
  • the anti-HIV/gp120 antibody described above was incubated on ice for 1 hour; one of the chamber slides was selected as the 0-hour control, rinsed once with precooled PBS, and then fixed with 2% paraformaldehyde; the other 4 slides were placed at 37°C for 0.5 hour, 2 hour, 4 hour, and 8 hour, respectively, rinsed once with precooled PBS, and fixed with 2% paraformaldehyde at 4°C overnight; rinsed twice with PBST (PBS containing 0.05% Tween-20), and 25 10 ⁇ L of fixation and permeabilization buffer (PBS containing 2% goat serum and 0.5% Triton X-100) was incubated at room temperature for 10 minutes; rinsed twice with PBST, and 250 ⁇ L of mouse anti-LAMP1 antibody (1:50 dilution, BD) and AF488-labeled goat anti-human IgG antibody (2 ⁇ g, Molecular Probes) diluted in FACS buffer (PBS containing 2% FBS)
  • HER2 The degradation of HER2 was detected by Western-blot method, which further verified that the anti-HER2 complementary bispecific antibody was internalized and transported into lysosomes for degradation after binding to HER2 on the cell surface.
  • the specific method is as follows: BT474 cells were collected, resuspended to 1 ⁇ 10 6 cells/mL with complete culture medium (RPMI-1640 culture medium containing 10% FBS), and the cell suspension was dispensed into 1.5-mL EP tubes at 500 ⁇ L/tube; the antibodies to be tested (including complementary bispecific antibody 04BS-1123-ST06, control antibody Trastuzumab, and monospecific antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 corresponding to the complementary bispecific antibodies) were diluted to 300 ⁇ g/mL with complete culture medium, and then added into the above EP tubes at 100 ⁇ L/tube (the final antibody concentration was 50 ⁇ g/mL); each EP tube was placed at 37°C/
  • the protein concentration in the extract is quantified using a BCA kit (Thermo); according to the protein concentration, the protein concentration in each extract is quantified using RIPA lysis buffer.
  • the protein was diluted to 0.125 ⁇ g/ ⁇ L, and then 5 ⁇ SDS-PAGE sample buffer containing DTT was added and heated at 95°C for 5 minutes to prepare SDS-PAGE loading samples.
  • the treated samples were loaded onto a 10% SDS-PAGE gel at 10 ⁇ L/lane (i.e., 1 ⁇ g/lane) for electrophoresis, and then the proteins were transferred to 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 the membrane was cut into two halves at the 55 kDa molecular weight marker, wherein the upper half was added with a 1:5000 dilution of rabbit anti-human HER2 antibody (CST) and incubated at 4°C overnight, and the lower half was added with a 1:10,000 dilution of mouse anti-human GAPDH antibody (Proteintech) and incubated at room temperature for 1 hour; after the incubation, it was washed 3 times with PBST, and then a 1:5000 dilution of goat anti-rabbit IgG-HRP antibody (Jackson Immuno) and a goat anti-mouse IgG-HRP antibody (Jack
  • This example also detects the effect of complementary bispecific antibody 04BS-1123-ST06 on BT474 cell proliferation.
  • the specific method is as follows: BT474 cells (10,000 cells/well) were inoculated in a 96-well white-walled plate, and gradient dilutions of the antibodies to be tested (including 04BS-1123-ST06, Trastuzumab, Pertuzumab, Hu2117-HK304-06, Hu2126-H2K1-L71-H72b-Mu14) were added, and the cells were incubated at 37°C/5% CO 2 for 5 days, and then the detection reagent CellCounting-Lite 2.0Luminescent Reagent (Nanjing Novizan) was added, and the chemiluminescence value was read with a multifunctional microplate reader (Varioskan, Thermo), and the experimental data were analyzed using GraphPad Prism 9 software.
  • the complementary bispecific antibody 04BS-1123-ST06 can effectively inhibit the proliferation of BT474 cells, but the corresponding monospecific antibodies have no activity in inhibiting the proliferation of BT474 cells. Since the proliferation of BT474 cells depends on the signal transduction mechanism activated by the homodimers formed by the overexpressed HER2 on the cell membrane, it is inferred that the cell proliferation inhibition activity of the complementary bispecific antibody is due to the endocytosis/degradation of the cell surface HER2, which prevents the formation of HER2 homodimers.
  • NanoBiT structural complementation reporter system was used to further verify whether the anti-HER2 complementary bispecific antibody would affect NRG-1-induced HER2/HER4 dimerization.
  • the NanoBiT system consists of an LgBiT subunit and a SmBiT subunit, which can be fused with the protein to be tested respectively; if the two proteins to be tested interact to form a dimer, LgBiT will complement the SmBiT structure to form a functional luciferase, which will then react with the substrate to produce a luminescent signal.
  • the DNA sequences encoding the extracellular and transmembrane regions of HER2 and HER4 were first inserted into the NanoBiT system pBiT1.3-C and pBiT2.3-C vectors by molecular cloning technology, and then the two plasmids were co-transfected into U2OS cells; the cells were collected 24 hours after transfection, plated in a 96-well white-walled plate, and cultured overnight at 37°C.
  • Complementary bispecific antibody 04BS-1123-ST06 humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14, chimeric antibodies mAb2117 and mAb2126, and control antibodies
  • Trastuzumab and Pertuzumab were diluted in series, and then mixed with NRG-1 at a volume ratio of 1:1 for standby 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 at this time was read as the background.
  • the control antibodies Trastuzumab and Pertuzumab can significantly inhibit NRG-1-induced HER2/HER4 dimerization, while the chimeric antibodies mAb2117 and mAb2126, the monospecific humanized antibodies Hu2117-HK304-06 and Hu2126-H2K1-L71-H72b-Mu14 ( Figure 15A), and the complementary bispecific antibody 04BS-1123-ST06 constructed based on these two monospecific humanized antibodies ( Figure 15B) have no inhibitory effect.
  • iPSC Induced Pluripotent Stem Cell
  • iPSC Induced Pluripotent Stem Cell
  • Related Culture kits were purchased from Fujiflim Cellular Dynamics. According to the method described in the instruction manual provided by the manufacturer, iPSC was plated into a 6-well plate and cultured at 37°C and 5% CO2 for one week to allow the cells to fully differentiate into human cardiomyocytes (iCell Cardiomyocytes).
  • the effect of the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 on NRG-1-induced AKT phosphorylation in human cardiomyocytes was detected according to the method described in Item 1.10 of Example 1.
  • 04BS-1123-ST06 had no inhibitory effect on NRG-1-induced AKT phosphorylation in human cardiomyocytes, indicating that the complementary bispecific antibody does not affect the regulation of NRG-1-induced HER2 dimerization and its downstream signal transduction pathways in cardiomyocytes.
  • the ForteBio test confirmed that the complementary bispecific antibody 04BS-1123-ST06 containing the TM mutation had no obvious binding reaction with Fc ⁇ RI, Fc ⁇ RIIa (167H), Fc ⁇ RIIb, Fc ⁇ RIIIa (176V), Fc ⁇ RIIIa (176F) and C1q, however, the binding affinity of the complementary bispecific antibody to FcRn was not significantly changed compared with Trastuzumab (data not shown).
  • this example further uses Jurkat cells (Promega) expressing the firefly luciferase reporter gene regulated by the Fc ⁇ RIIIa-176V receptor and the NFAT response element as effector cells, BT474 cells overexpressing HER2 as target cells, and Trastuzumab as a positive control antibody to further verify whether the complementary bispecific antibody 04BS-1123-ST06 containing the TM mutation has ADCC activity.
  • the specific method is as follows: BT474 cells are collected by centrifugation, resuspended in ADCC detection buffer (RPMI-1640 culture medium containing 0.5% FBS), and added to a 96-well white-walled plate at 1.5 ⁇ 10 4 cells/well.
  • the antibody to be tested is diluted in a gradient manner with ADCC detection buffer, and 25 ⁇ L/well is added In the above 96-well plate. After collecting the effector cells by centrifugation and resuspending them with ADCC detection buffer, 1.5 ⁇ 10 5 cells/well were added to the above 96-well plate, and then incubated overnight at 37°C and 5% CO 2. The next day, the 96-well plate was placed at room temperature for equilibrium, and then the detection reagent Bio-Lite Luciferase Reagent (Nanjing Novogene) was added, and the chemiluminescence value was read using a multifunctional microplate reader (Varioskan, Thermo), and the experimental data was analyzed using GraphPad Prism 9 software.
  • test results are shown in Figure 17. Unlike the positive control antibody Trastuzumab, 04BS-1123-ST06 did not induce any luciferase expression even at the highest concentration, indicating that the anti-HER2 complementary bispecific antibody with the TM mutation introduced in the Fc region does not have ADCC activity.
  • the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 is coupled to a small molecule toxin compound Eribulin with anti-tumor activity through a cleavable linker (e.g., Mal-(PEG) 2 -VC-PAB, Mal-(PEG) 2 -GGFG) to prepare an anti-HER2 complementary bispecific ADC.
  • a cleavable linker e.g., Mal-(PEG) 2 -VC-PAB, Mal-(PEG) 2 -GGFG
  • the Eribulin is prepared according to the method described in ZL201910197071.8 or ZL201910509222.9, and WO1999065894.
  • the preparation method of the drug payload Mal-(PEG) 2 -GGFG-Eribulin is as follows: NH 2 -(PEG) 2 -COOH and maleic anhydride are added to acetic acid, heated to reflux overnight, acetic acid is removed by rotary evaporation, and the residue is prepared by HPLC and freeze-dried to obtain Mal-(PEG) 2 -COOH.
  • 2Cl Trt Resin is used as a solid phase carrier, and Fmoc protection is removed by using 20% piperidine/DMF (v/v) solution, and then HOBT/DIC is used as a condensation system and DMF is used as a reaction solvent.
  • Fmoc-GGFG-OH and Eribulin were placed in a single-mouth bottle, DMF was added to dissolve, the temperature was lowered to 0°C, DIPEA (161.3 mg, 1.25 mmol) and DECP (122.3 mg, 0.75 mmol) were added, and the mixture was reacted at room temperature and monitored by HPLC. After the reaction was completed, 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 the crude Fmoc-GGFG-Eribulin.
  • the crude Fmoc-GGFG-Eribulin was placed in a single-mouth bottle, THF was added, the temperature was lowered to 0°C, two equivalents of LiOH aqueous solution were added, the mixture was reacted at room temperature and monitored by HPLC. After the reaction was completed, the pH was adjusted to about 6-7 with 50% acetic acid, THF was removed by rotary evaporation, and then purified by high-performance liquid chromatography to collect the pure NH 2 -GGFG-Eribulin. HATU was added to a DMF solution of Mal-(PEG) 2 -COOH and sodium bicarbonate at room temperature.
  • the preparation method of the anti-HER2 complementary bispecific ADC is as follows: ZnCl 2 (3.0 mM) and TCEP (6.0 mM) are slowly added to the anti-HER2 complementary bispecific antibody 04BS-1123-ST06 (1.5 mM) solution (the antibody preparation buffer is 50 mM Histidine-HCl, 8% sucrose, pH 5.5) under an ice water bath, wherein the final reaction concentrations of ZnCl 2 , TCEP and 04BS-1123-ST06 antibody are 0.10 mM, 0.20 mM and 0.05 mM, respectively. After mixing, the mixture is reduced by shaking at 8° C. for about 16 hours.
  • Mal-(PEG) 2 -VC-PAB-Eribulin or Mal-(PEG) 2 -GGFG-Eribulin dissolved in DMSO was added to a final concentration of 0.32 mM under ice-water bath, and after continuous reaction at 8°C for 3 hours, cysteine was added to a final concentration of 0.05 mM to deplete excess Mal-(PEG) 2 -VC-PAB-Eribulin or Mal-(PEG) 2 -GGFG-Eribulin.
  • EDTA was added to a final concentration of 0.15 mM to chelate Zn 2+ , and related impurities were removed using Pellicon XL Ultrafiltration membrane filter (Module Ultracel 30 kDa/0.005 m 2 , purchased from Merck).
  • Pellicon XL Ultrafiltration membrane filter Module Ultracel 30 kDa/0.005 m 2 , purchased from Merck.
  • HIC-HPLC BioCore HIC-Butyl 5 ⁇ m/4.6 ⁇ 100 mm, purchased from NanoChrom
  • SEC-HPLC Zenix-C SEC-300, purchased from Cypermethrin
  • AUC +1 is the area under the peak curve corresponding to the ADC coupled with one small molecule compound
  • AUC +2 is the area under the peak curve corresponding to the ADC coupled with two small molecule compounds
  • ⁇ AUC total is the area under all peak curves.
  • the average DAR value of the ADC prepared in this example is 4.0 ⁇ 0.5, and the purity is>95%.
  • the binding activity of the exemplary anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) to the HER2 recombinant protein and the cross-binding activity with other members of the human ErbB/HER family were detected by ELISA method, and the antigens included human HER2 extracellular region recombinant protein and human EGFR, HER3 and HER4 extracellular region recombinant protein.
  • the test results are shown in Figure 18, and the anti-HER2 complementary bispecific ADC (ST06-VCP-Eribulin) can specifically bind to HER2, but has no cross-binding activity with other members of the human ErbB/HER family.
  • the antigen binding specificity of the anti-HER2 complementary bispecific ADC is equivalent to that of its corresponding naked anti-04BS-1123-ST06 (data not shown), indicating that the antigen binding specificity of the complementary bispecific antibody is not changed after coupling with a small molecule toxin compound.
  • HER2 expression levels and drug resistance of the tumor cell lines used are shown in Table 13; JIMT-1 is HER2 SKOV-3R* is a cell line that overexpresses HER2 and has acquired resistance to DS-8201.
  • in vitro tumor cell killing activity detection The specific steps of in vitro tumor cell killing activity detection are as follows: collect each cell by trypsin digestion, resuspend the cells in a medium containing 10% FBS, and then inoculate them in a 96-well white wall plate, with 5,000 or 10,000 cells per well. The cells were cultured in a 37°C, 5% CO 2 incubator. After the cells were completely attached to the wall, ST06-VCP-Eribulin, ST06-GGFG-Eribulin or DS-8201 diluted in a gradient in the corresponding culture medium was added.
  • the cell culture plate was removed from the incubator after incubation at 37°C/5% CO 2 for 3 to 5 days, and CellCount Lite2.0 reagent (Nanjing Novozyme) was added after equilibration to room temperature, and the cells were shaken and mixed for 2 to 5 minutes to fully lyse the cells; after standing at room temperature for 10 minutes, the chemiluminescence value was read using a multi-function microplate reader (Varioskan, Thermo). Data were analyzed using GraphPad Prism 9 software and expressed as a percentage of the chemiluminescence value relative to untreated control wells.
  • test results are shown in Figure 19 and Table 13.
  • the anti-HER2 complementary bispecific ADC including ST06-GGFG-Eribulin and ST06-VCP-Eribulin
  • the benchmark molecule DS-8201 both showed strong cytotoxicity, and the cell killing activity of the complementary bispecific ADC was significantly higher than that of DS-8201.
  • the cell killing activity of the complementary bispecific ADC was significantly higher than that of DS-8201; in the HER2-low-expressing tumor cell line ZR-75-1, the complementary bispecific ADC also showed effective cell killing activity, while DS-8201 had almost no cell killing activity.
  • the complementary bispecific ADC In tumor cell lines T47D, RT-112, MCF-7 expressing lower levels of HER2, and tumor cell line MDA-MB-468 without HER2 expression, the complementary bispecific ADC has no obvious cytotoxicity, indicating that the complementary bispecific ADC has a good safety window.
  • the complementary bispecific ADC (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) also showed significant cytotoxicity.
  • the anti-HER2 complementary bispecific ADC has a broader spectrum of cell killing activity, which can not only kill tumor cells with overexpression of HER2, but also directly kill tumor cells with low expression of HER2, and also has a killing effect on tumor cells that do not respond to DS-8201.
  • this example screened and obtained a tumor cell line (named SKOV-3R*) with acquired resistance to DS-8201 by continuously subculturing SKOV-3 cells overexpressing HER2 in a medium containing gradually increasing concentrations of DS-8201.
  • the cell line can proliferate normally in a medium containing 100nM DS-8201.
  • the results of the in vitro cell killing experiment showed that the complementary bispecific ADC (including ST06-GGFG-Eribulin and ST06-VCP-Eribulin) can effectively kill the acquired resistance cell line SKOV-3R*.
  • ST06-GGFG-Eribulin can cause significant cell killing after 3 days of BT474 cells, and MDA-MB-468 cells cultured with its culture supernatant also showed significant cell death, while the freshly prepared ADC solution had no killing activity on the cells, indicating that while ST06-GGFG-Eribulin kills BT474 cells, the small molecule toxin compound Eribulin released into the culture medium can kill MDA-MB-468 cells that do not express HER2.
  • this example further verifies the bystander killing effect of ST06-GGFG-Eribulin by co-culturing BT474 cells and Jurkat cells that do not express HER2.
  • the specific method is as follows: BT474 cells and Jurkat cells expressing GFP are collected and plated into a 24-well cell culture plate at 1 ⁇ 10 5 cells/well, respectively, either alone or together, and then ST06-GGFG-Eribulin is added at a final concentration of 1 nM. After culturing at 37° C.
  • the cells in each well are collected into a flow tube, and the supernatant is removed by centrifugation, and the cells are resuspended in 100 ⁇ L FACS buffer (PBS containing 1% BSA), and 3 ⁇ L 7-AAD is added for dead cell staining and then flow analysis is performed. Among them, 10 ⁇ L of cell fluid was collected and analyzed in each tube. After excluding 7-AAD-positive dead cells, BT474 and Jurkat cells were distinguished using the side scattered light channel and the GFP channel.
  • This example detects the anti-tumor activity of anti-HER2 complementary bispecific ADC in three mouse subcutaneous xenograft tumor models constructed based on human tumor cell lines, wherein the reference control drug is DS-8201 (trade name Enhertu, purchased from Daiichi Sankyo Pharmaceutical); the three tumor models are: NCI-N87 gastric cancer tumor model with overexpression of HER2, JIMT-1 breast cancer tumor model with moderate expression of HER2, and RT-112 bladder cancer tumor model with low expression of HER2.
  • the specific method is as follows: After the cell lines (NCI-N87, JIMT-1, and RT-112) are cultured to the logarithmic growth phase, the cells are collected and inoculated into the subcutaneous dorsal area of the right forelimb of immunodeficient mice (Nude or SCID) at 5 to 10 ⁇ 10 6 cells per mouse.
  • ST06-GGFG-Eribulin showed an anti-tumor activity comparable to that of DS-8201 in the HER2-overexpressed NCI-N87 tumor model, but showed a stronger anti-tumor activity than DS-8201 in the JIMT-1 tumor model with moderate HER2 expression, and the difference was statistically significant (p ⁇ 0.01).
  • ST06-GGFG-Eribulin showed significantly superior antitumor activity to DS-8201 at an equivalent DAR dose (9 mg/kg), and the difference in antitumor activity was statistically significant (p ⁇ 0.01).
  • no weight loss or other toxic side effects were observed in tumor-bearing mice (data not shown).
  • this example uses the NCI-N87 tumor cell line to construct a mouse xenograft tumor model with acquired resistance to DS-8201.
  • the construction method is as follows: the wild-type NCI-N87 cells are continuously subcultured in a culture medium containing gradually increasing concentrations of DS-8201, and a cell line subpopulation with a certain resistance to DS-8201 is screened and named NCI-N87(R).
  • NCI-N87(R) cells were expanded and cultured in RPMI-1640 medium containing 10% serum until the exponential growth phase, the cells were collected and resuspended in a mixture of PBS and Matrigel (volume ratio of 1:1), and then 1 ⁇ 10 7 cells were subcutaneously inoculated on the back of the right forelimb of each nude mouse (BALB/c Nude), with 5 female nude mice inoculated each time, and in vivo screening of acquired drug-resistant tumors was carried out.
  • the specific screening steps are as follows: when the tumor volume of the mouse reaches 200 ⁇ 50mm 3 , DS-8201 (at a dose of 3 mg/kg) is injected into the tail vein once a week to screen tumor-bearing nude mice whose tumors still grow stably after administration; the number of administrations is 3 to 5 times, depending on the growth rate of the tumor. After the tumor volume grew to 900-1000 mm3 , the mice were euthanized and the tumors were collected. After removing the necrotic tissue, the tumors were cut into small pieces of about 30 mm3 and inoculated subcutaneously on the right side of the back of 5 female nude mice near the forelimbs to carry out the next round of DS-8201 administration and screening.
  • the tumors of the five tumor-bearing nude mice could grow stably after the administration of DS-8201 (at a dose of 3 mg/kg), and the growth trend was relatively consistent.
  • the tumor volume grew to about 1000 mm3
  • the mice were randomly selected. Two mice were euthanized, and the tumors were collected and the necrotic tissues were removed. The tumors were cut into small pieces of about 30 mm3 and then inoculated subcutaneously on the right side of the back near the forelimb of 50 female nude mice.
  • mice with relatively uniform tumor growth and an average volume of about 210 mm 3 were randomly divided into two ST06-GGFG-Eribulin test groups (divided into 3 mg/kg group and 10 mg/kg group according to the dosage), one DS-8201 control group (dosage of 3 mg/kg) and one vehicle control group, wherein the ST06-GGFG-Eribulin test group with a dosage of 3 mg/kg, the DS-8201 control group and the vehicle control group each had 8 mice, and the ST06-GGFG-Eribulin test group with a dosage of 10 mg/kg had 5 mice.
  • the experimental results are shown in Figure 22.
  • the mouse tumor continued to grow stably after the administration of DS-8201, indicating that the modeling was successful, that is, the tumor model was indeed a xenograft tumor model with acquired resistance to DS-8201.
  • ST06-GGFG-Eribulin showed anti-tumor activity at doses of 3 mg/kg and 10 mg/kg; especially at a dose of 10 mg/kg (equivalent DAR), the tumor almost completely regressed after the administration of ST06-GGFG-Eribulin, indicating that the complementary bispecific ADC (ST06-GGFG-Eribulin) can break through the acquired resistance of DS-8201 and has potential efficacy for DS-8201 relapsed/refractory tumors.
  • ST06-GGFG-Eribulin complementary bispecific ADC

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供一种抗HER2抗体或其抗原结合片段、由其构建的抗HER2互补性双特异抗体和互补性双特异抗体-药物偶联物(ADC)、以及包含它们的药物组合物和药盒。还提供所述抗体、ADC、药物组合物和药盒用于治疗与HER2异常表达相关联的疾病的方法。

Description

抗HER2互补性双特异抗体-药物偶联物及其制备方法和用途 技术领域
本发明涉及抗HER2抗体或其抗原结合片段，以及基于此而构建的抗HER2互补性双特异抗体和包含该类抗体的抗体-药物偶联物。本发明还提供了制备该互补性双特异抗体及其抗体-药物偶联物的方法，以及用于治疗与HER2异常表达相关联的疾病的方法。
背景技术
人表皮生长因子受体2(Human Epidermal Growth Factor Receptor 2，简称HER2或ErbB2)是受体酪氨酸激酶ErbB/HER家族成员之一。ErbB/HER家族受体通过二聚化而激活其介导的下游信号转导通路，并由此参与细胞生长、分化和存活的调控。HER2是分子量约为185kDa的跨膜糖蛋白，它由胞外结构域(Extracellular Domain)、跨膜结构域(Transmembrane Domain)和胞内酪氨酸激酶结构域(Intracellular Tyrosine Kinase Domain)组成。ErbB2/HER2基因突变和/或基因扩增会导致HER2过表达(Overexpression)，其在IHC及FISH检测中表现为IHC 3+或IHC 2+/FISH+。临床研究显示HER2在多种癌症(例如乳腺癌、卵巢癌、子宫内膜癌、胃癌、前列腺癌、肺癌等)中过表达，且该过表达与肿瘤的侵袭性和不良预后有关联。
多款靶向HER2的治疗性药物已获批用于治疗HER2过表达的乳腺癌和胃癌，这些药物显著延长了患者的生存期；但是，仍有相当一部分的癌症患者对这些疗法不响应。除此之外，现有的HER2靶向疗法还存在以下缺陷而限制了其临床疗效并导致安全性风险：
(1)现有的HER2靶向疗法对HER2表达量相对较低的肿瘤细胞缺乏有效杀伤力，所述HER2表达量相对较低是指在IHC检测中表现为2+/FISH-或IHC 1+(定义为“HER2低表达”)；由于HER2的表达量在HER2过表达肿瘤中具有高度异质性，因此所述肿瘤中HER2低表达的肿瘤细胞很容易对现有疗法产生耐药性，导致大多数癌症患者在接受现有HER2靶向疗法6～12月后会因耐药性而发生疾病进展；
(2)相对于HER2过表达，约70％的乳腺癌病人表征为HER2低表达，针对这一患病人群，DS-8201是目前唯一获批的HER2靶向药物，但其在三期临床试验DESTINY-Breast04中也仅有约50％的应答率，并且在试验期间与药物相关的间质性肺病/肺炎的发生率为12.1％(Modi等,N Engl J Med 2022,387:9-20)，突显现有靶向HER2疗法在治疗HER2低表达肿瘤的有效性和安全性方面存在极大的提升空间；
(3)HER2介导的信号转导通路对于维持心肌细胞的生存、修复心肌细胞的损伤、以及保持心肌细胞功能的完善至关重要；目前获批的抗HER2抗体、ADC或小分子抑制剂都能够阻断或者扰乱HER2信号转导通路，因此均具有诱发心脏功能障碍的毒副作用(Cardiotoxicity)，例如Trastuzumab和Pertuzumab在治疗中均可能引发左心室射血分数(LVEF)降低和充血性心脏衰竭，尤其是Trastuzumab在与蒽环类药物同时给药时诱发心脏衰竭的风险更高，这导致在应用这些药物进行治疗前需要严格筛选适应症人群或对患者进行预防性心脏保护治疗。
因此，本领域仍存在较大的未满足的临床需求，亟需开发一种更加安全(尤其是解决现有靶向HER2疗法的心脏毒性问题)，并且对HER2低表达的肿瘤细胞具有更强杀伤活性的靶向药物以破解现有疗法的安全性及耐药性问题。
发明内容
本发明提供了一种靶向HER2的互补性双特异抗体-药物偶联物(HER2-targeting Biparatopic ADC)。一方面，所述互补性双特异ADC能够特异性地结合HER2胞外结构域(也称“胞外区”)中的两个不同表位，因此能够有效地交联(Crosslink)细胞表面HER2以形成矩阵状多聚体，进而诱发快速、高效内吞，并引导内吞后的胞内体(Endosome)由通常的重循环转运途径(Recycling)转变为溶酶体转运途径(Lysosomal Trafficking)，由此使得结合于细胞表面的ADC被高效转运(几乎100％)进入溶酶体发生降解，从而释放出更多的小分子毒素至细胞质中以发挥细胞毒性以及后续的旁观者杀伤效应。因此，所述互补性双特异ADC具有更广谱的靶向杀伤活性，其不仅能杀伤HER2过表达的癌细胞，也能够直接杀伤HER2低表达(IHC 2+/FISH-或IHC 1+)的癌细胞，由此极大地降低了因HER2表达的异质性而产生耐药性的几率；另一方面，所述互补性双特异ADC的抗原结合表位不同于Trastuzumab或Pertuzumab，其不会干扰HER2的二聚化或影响HER2下游信号转导通路的调控，因此不会影响HER2在心肌细胞中的正常生物学功能。
一方面，本发明提供了一种分离的抗HER2抗体或其抗原结合片段，所述的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外结构域，对表达HER2的肿瘤细胞具有较高的结合活性或结合亲和力(例如结合亲和力常数[KD]值＜1×10-8M、＜5×10-9M、或＜1×10-9M)，以及不与ErbB/HER家族其它成员(包括EGFR、HER3和HER4)发生交叉结合反应。所述抗HER2抗体或其抗原结合片段对HER2及其下游信号转导通路不具有抑制或激活作用。
在一些实施方案中，所述抗HER2抗体包括鼠源抗HER2抗体或其衍生的嵌合抗体和/或人源化抗体及其优化抗体。在一些实施方案中，所述的抗HER2抗体或其抗原结合片段特异性地结合HER2胞外区的亚结构域(Subdomain)1、3和/或4，优选HER2胞外区亚结构域1或3。在一些实施方案中，用于构建抗HER2互补性双特异抗体的两种抗HER2抗体或其抗原结合片段分别特异性地识别HER2胞外区中的不同表位，且彼此之间不存在竞争性结合关系。
一方面，本发明提供了一种抗HER2互补性双特异抗体，其包含第一抗原结合结构域和第二抗原结合结构域，所述第一和第二抗原结合结构域特异性地结合HER2的不同表位，导致所述双特异性抗体能够在细胞表面交联HER2形成簇(Cluster)，并且簇的形成受制于细胞表面HER2的表达丰度；簇的形成会引发其被细胞快速内吞并被转运至溶酶体中降解。相较于本发明所述的单特异性抗HER2抗体和/或Trastuzumab，所述互补性双特异抗体具有显著提高了的HER2内化率以及更高的溶酶体转运/降解效率。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一和第二抗原结合结构域特异性地结合HER2的不同(非重叠或非竞争)表位，所述表位包括位于HER2胞外区亚结构域1、3和/或4中的序列。在一个实施方案中，所述第一抗原结合结构域特异性地结合HER2胞外区亚结构域3，所述第二抗原结合结构域特异性结合HER2胞外区亚结构域1，所结合的表位均不同于Trastuzumab或Pertuzumab的抗原结合表位。在一个实施方案中，所述互补性双特异抗体对细胞表面HER2及其下游信号转导通路不具有抑制或激活作用。在一个实施方案中，所述互补性双特异抗体在结合并交联细胞表面的HER2后引发其被细胞快速内化以及高效的溶酶体转运。在一个实施方案中，所述互补性双特异抗体能够诱导细胞表面HER2降解。在一个实施方案中，所述互补性双特异抗体通过交联细胞表面HER2，使其内化并转运至溶酶体内降解，导致细胞表面HER2的丰度显著降低，因而有效抑制HER2过表达肿瘤细胞的增殖。
一方面，本发明提供了一种ADC，其将小分子毒素化合物通过接头(Linker)偶联至本发明的抗HER2互补性双特异抗体。所述ADC能够特异性地结合/交联表达于肿瘤细胞表面的HER2而聚集成“簇”，使得ADC被快速内吞并转运至溶酶体内降解，显著提高了ADC中的小分子毒素化合物进入肿瘤细胞和在肿瘤细胞内释放的效率，因此所述互补性双特异ADC相较于T-DM1和/或DS-8201具有更广谱的肿瘤细胞杀伤活性，即所述ADC对HER2过表达和HER2低表达的肿瘤细胞均具有直接杀伤活性。所述ADC可以由式(Ⅰ)表示：
Ab-(L-D)p  (Ⅰ)
其中Ab表示本发明的抗HER2互补性双特异抗体；
D表示小分子毒素化合物(Drug)；
L表示将Ab偶联至D的可裂解接头(Cleavable Linker)；以及
p表示偶联至Ab上的(L-D)的拷贝数，为2至8。
在一些实施方案中，所述小分子毒素化合物包括细胞毒素和化疗药物。在一个实施方案中，所述小分子毒素化合物是细胞毒素，包括微管蛋白抑制剂和DNA损伤剂；优选地，所述微管蛋白抑制剂包括艾日布林(Eribulin)、奥瑞他汀(Auristatins)类衍生物(例如，MMAE、MMAF、MMAD)、Tubulysins、Cryptomycins和美登素(Maytansinoids)类衍生物(例如，DM1、DM2、DM3、DM4)，所述DNA损伤剂包括拓扑异构酶抑制剂(例如，喜树碱类衍生物SN-38、依沙替康[Exatecan]和DXd[Exatecan derivative for ADC])、吡咯并苯并二氮杂卓(PBD)、卡奇霉素(Calicheamicin)及其衍生物(例如N-乙酰基卡奇霉素[CMC])、和倍癌霉素(Duocarmycin)。在一个实施方案中，所述小分子毒素化合物为艾日布林。
在一些实施方案中，所述可裂解接头可以为包含可裂解部分的任何接头，所述可裂解部分包含可裂解的任何化学键。在一些具体实施方案中，所述可裂解接头包含可裂解肽部分，其能够被细胞内肽酶或蛋白酶裂解，所述可裂解肽部分包含氨基酸单元，所述氨基酸单元包含二肽、三肽、或四肽。在一些具体实施方案中，所述可裂解接头可以包含至少一种将本发明抗HER2互补性双特异抗体(Ab)缀合于小分子毒素化合物(D)的间隔区(Spacer)，所述间隔区包含与抗体缀合的间隔区，和/或与小分子毒素化合物缀合的第二间隔区。在一些实施方式中，所述与抗体缀合的间隔区是亲水性的，示例性的间隔区包含聚乙二醇(PEG)。在一些实施例中，所述间隔区经由丁烯二酰亚胺(Mal)连接于本发明的抗HER2互补性双特异抗体。在一些实施方式中，所述与小分子毒素化合物缀合的第二间隔区用于将可裂解接头的可裂解部分(例如可裂解肽)连接于小分子毒素化合物。在一些实施例中，所述与小分子毒素化合物缀合的第二间隔区具有自焚碎裂(Self-immolation)的特性，所述自焚碎裂间隔区包含对氨基苯甲基单元。在一些具体实施方案中，所述可裂解接头包含Mal-间隔区和可裂解肽部分。在一些具体实施方案中，所述可裂解接头的Mal-间隔区与本发明ADC中的抗体部分的一个或多个氨基酸残基偶联。在一些实施方案中，所述Mal-间隔区的顺丁烯二酰亚胺基可以与所述抗体的恒定区和/或可变区中特定位置处的半胱氨酸残基的巯基反应。在一些具体实施方案中，所述可裂解接头的第二间隔区偶联至本发明ADC中的小分子毒素化合物部分，所述小分子毒素化合物是艾日布林或其衍生物。在一些具体实施方案中，所述可裂解接头的可裂解部分(例如可裂解肽)可以直接缀合于所述ADC的小分子毒素化合物部分，所述小分子毒素化合物是艾日布林或 其衍生物。
在一些实施方案中，p为2至8，例如4至8。在本文中，p可为大于0的整数或非整数。
在另一个方面，本发明涉及编码抗HER2互补性双特异抗体及其对应的单特异性抗体或其抗原结合片段的分离的核酸分子(也称为“多核苷酸”)，以及包含所述核酸的表达载体和包含核酸或表达载体的宿主细胞。本发明还涉及使用所述宿主细胞制备本文所述单特异性抗HER2抗体或其抗原结合片段以及抗HER2互补性双特异抗体的方法，所述方法包括培养所述宿主细胞并从培养基中回收所述抗体或其抗原结合片段。
在另一个方面，本发明涉及一种药物组合物，其包含本文所述的单特异性抗HER2抗体或其抗原结合片段，或抗HER2互补性双特异抗体，或抗HER2互补性双特异抗体-药物偶联物，以及药学上可接受的载体。
在另一个方面，本发明涉及一种药盒，所述药盒包含有效量的本发明所述的抗HER2单特异性抗体或其抗原结合片段、抗HER2互补性双特异抗体、抗HER2互补性双特异抗体-药物偶联物、或药物组合物，以及任选的至少一种另外的肿瘤治疗剂。
在另一个方面，本发明涉及一种对受试者中的表达HER2的肿瘤进行治疗的方法，所述方法包括向有需要的受试者施用本发明所述的抗HER2互补性双特异抗体、抗HER2互补性双特异抗体-药物偶联物、药物组合物或药盒。或者，本发明涉及本文所述的抗HER2互补性双特异抗体、抗HER2互补性双特异抗体-药物偶联物、药物组合物或药盒在用于制备对受试者中的表达HER2的肿瘤进行治疗的药物中的用途。或者，本发明涉及用于对受试者中的表达HER2的肿瘤进行治疗的本文所述的抗HER2互补性双特异抗体、抗HER2互补性双特异抗体-药物偶联物、药物组合物或药盒。在一些实施方案中，所述肿瘤包括HER2过表达(IHC 3+、或IHC 2+/FISH+)的肿瘤和/或HER2低表达(IHC 2+/FISH-、或IHC 1+)的肿瘤。在一些实施方案中，所述抗HER2互补性双特异抗体-药物偶联物或其药物组合物对HER2过表达和/或HER2低表达的肿瘤细胞均具有杀伤作用。在一些实施方案中，所述受试者为哺乳动物。在一些实施方案中，所述肿瘤包括乳腺癌、卵巢癌、宫颈癌、结直肠癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、黑色素瘤、胰腺癌、肝癌、胆管癌、肾癌、膀胱癌、甲状腺癌、前列腺癌、子宫内膜癌等，所述肿瘤可以是HER2过表达或低表达的。
在一些实施方案中，所述肿瘤为对现有HER2靶向治疗剂产生耐药性的肿瘤。在一些实施方案中，所述肿瘤为对包括Trastuzumab、Pertuzumab、T-DM1和/或DS-8201的HER2靶向治疗剂具有耐药性的肿瘤。在一个实施方案中，所述肿瘤为对包括Trastuzumab、Pertuzumab、T-DM1、DS-8201和/或紫杉烷类(例如，Paclitaxel、Docetaxel、Cabazitaxel等)的靶向治疗剂不响应或响应较差的肿瘤。
在一些实施方案中，所述受试者包括不适合接受现有HER2靶向疗法或难治疗、或在接受现有HER2靶向疗法后产生耐药性或复发的患者。在一些实施方案中，所述的现有HER2靶向疗法包括使用Trastuzumab、Pertuzumab、T-DM1、和/或DS-8201进行治疗。
在另一个方面，本发明涉及一种检测和/或测量样品中的HER2或表达HER2的肿瘤细胞的方法，或者筛选对本发明所述的抗HER2互补性双特异ADC治疗有响应的癌症患者的方法，包括将本发明所述的抗HER2单特异性抗体或其抗原结合片段、或抗HER2互补性双特异抗体与所述样品或分离自所述患者的生物样本进行孵育，检测所述抗体是否结合至所述样品或生物样本。
通过下面的附图和具体实施方案进一步说明本发明，本发明所公开的其它特征和优点将是显而易见，并且这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。在本发明中引用的所有参考文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。
附图说明
图1.利用ELISA检测各抗HER2嵌合抗体与人源ErbB/HER家族蛋白EGFR、HER2、HER3和HER4的结合活性，其中对照抗体包括Trastuzumab、Cetuximab、Patritumab。
图2.流式细胞术检测抗HER2嵌合抗体与过表达HER2的NCI-N87细胞的结合活性，其中对照抗体为Trastuzumab。
图3.人-鼠嵌合的HER2胞外区重组蛋白结构示意图(图3A)，以及利用ELISA方法测定抗HER2嵌合抗体的抗原结合表位所在的HER2胞外区亚结构域(图3B)。
图4.利用竞争ELISA方法检测各抗HER2嵌合抗体之间或与对照抗体Trastuzumab之间是否存在竞争结合关系，其中图4A为生物素标记的mAb2164分别与mAb2117、mAb2126、mAb2170和Trastuzumab竞争结合HER2重组蛋白的结果，图4B是mAb2117与生物素标记的mAb2128或生物素标记的mAb2126竞争结合HER2重组蛋白，以及mAb2164与生物素标记的mAb2128竞争结合HER2重组蛋白的结果。
图5.抗HER2嵌合抗体mAb2117和mAb2126对SKBR-3细胞中HER2胞内结构域Y1248位点磷酸化的作用，其中对照抗体包括Trastuzumab和Pertuzumab。
图6.抗HER2嵌合抗体mAb2117和mAb2126对NRG-1诱导的T47D细胞内AKT磷酸化的影响，其中对照抗体包括Trastuzumab和Pertuzumab。
图7.利用流式细胞术检测含有单点/多点突变的抗HER2单链抗体优化分子分别与BT474细胞(图7A)和RT-112细胞(图7B)的结合活性，其中对照抗体为母本单链抗体Hu2117HK。
图8.利用流式细胞术检测含有组合突变的抗HER2单链抗体优化分子(Hu2117-HK203、Hu2117-HK303、Hu2117-HK304、Hu2117-HK309、Hu2117-HK310)分别与BT474细胞和RT-112细胞的结合活性，其中对照抗体为母本单链抗体Hu2117HK。
图9.示例性抗HER2互补性双特异抗体04BS-109-WT的结构示意图。所述示例性互补性双特异抗体的构型为DVD-IgG结构，其中Fv结构域为Hu2117-HK304的可变区序列，IgG结构域为Hu2126-H2K1-L71-H72b-Mu14的全长序列，Fv结构域重链可变区与IgG结构域的重链通过连接子(G4S)1相连，Fv结构域轻链可变区与IgG结构域的轻链通过连接子(G4S)3相连，抗体重链Fc区含有L234F/L235E/P331S(EU Numbering)突变。
图10.利用流式细胞术测定抗HER2人源化抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14(简称Hu2126-7172b-Mu14)与BT474细胞的共结合或竞争结合关系。
图11.利用流式细胞术检测抗HER2互补性双特异抗体04BS-109-WT及其突变体分子在表达不同水平HER2的细胞株(BT474、JIMT-1、RT-112)中的内吞反应，其中对照抗体包括Trastuzumab、以及所述互补性双特异抗体04BS-1123-ST06对应的单特异性抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14(简称Hu2126-7172b-Mu14)。
图12.利用共聚焦显微镜方法检测抗HER2互补性双特异抗体04BS-1123-ST06在被SKBR-3细胞内吞后的溶酶体转运效率，其中对照抗体包括Trastuzumab和人源IgG同型抗体；图中箭头所指处为抗体的荧光信号位点与溶酶体的荧光信号位点相互重合，代表所述互补性双特异抗体与HER2所形成的交联聚集体或簇被细胞内吞并转运入溶酶体内；T代表抗体孵育时间(小时)。
图13.利用Western-blot方法检测抗HER2互补性双特异抗体04BS-1123-ST06引起的BT474细胞中HER2蛋白的降解反应，其中对照抗体包括Trastuzumab以及所述互补性双特异性抗体对应的单特异性抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14(简称Hu2126-7172b-Mu14)。
图14.抗HER2互补性双特异抗体04BS-1123-ST06对BT474细胞体外增殖的影响，其中对照抗体包括Trastuzumab、Pertuzumab、以及所述互补性双特异性抗体对应的单特异性抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14(简称Hu2126-7172b-Mu14)。
图15.利用报告基因法检测抗HER2单特异性抗体(图15A)和抗HER2互补性双特异抗体(图15B)对NRG-1诱导的HER2/HER4二聚化的影响，其中对照抗体包括Trastuzumab和Pertuzumab。
图16.利用Western-blot方法检测抗HER2互补性双特异抗体04BS-1123-ST06对NRG-1诱导的人心肌细胞内AKT磷酸化的影响，其中对照抗体包括Trastuzumab和Pertuzumab。
图17.利用报告基因法检测抗HER2互补性双特异抗体04BS-1123-ST06的ADCC活性，其中阳性对照抗体为Trastuzumab。
图18.利用ELISA方法检测抗HER2互补性双特异ADC(ST06-VCP-Eribulin)与人源ErbB/HER家族成员(包括EGFR、HER2、HER3和HER4)的结合特异性。
图19.利用体外细胞杀伤实验检测抗HER2互补性双特异ADC(包括ST06-GGFG-Eribulin和ST06-VCP-Eribulin)以及对标分子DS-8201在表达不同水平HER2的肿瘤细胞株中的增殖抑制活性。
图20.通过体外细胞杀伤实验检测抗HER2互补性双特异ADC的旁观者效应，其中图20A展示的结果为在ST06-GGFG-Eribulin处理BT474细胞3天后，检测其对BT474细胞的杀伤活性，同时收集培养上清(BT474-conditioned Medium)用于孵育MDA-MB-468细胞，并于3天后检测MDA-MB-468细胞的存活情况以检测所述ADC的旁观者杀伤效应，其中对照组为同步进行的用新鲜配制的ST06-GGFG-Eribulin孵育的MDA-MB-468细胞的存活情况。图20B为利用流式细胞术检测过表达HER2的BT474细胞与不表达HER2的Jurkat细胞在各自单独培养或共培养情况下的ST06-GGFG-Eribulin的细胞杀伤活性，图中左上角和右下角显示的数字分别是BT474细胞和Jurkat细胞的存活细胞计数。
图21.在基于肿瘤细胞株NCI-N87(图21A)、JIMT-1(图21B)和RT-112(图21C)而构建的小鼠皮下异种移植肿瘤模型中检测抗HER2互补性双特异ADC(ST06-GGFG-Eribulin)的体内抑瘤活性，其中对照组包括抗体和小分子化合物的混合物(ADMix)组、DS-8201组和溶媒组(Vehicle)，所有荷瘤小鼠均通过尾静脉注射给药，图中箭头表示给药时间点。
图22.利用对DS-8201具有后天获得型耐药性的小鼠皮下异种移植肿瘤模型检测抗HER2互补性双特异ADC(ST06-GGFG-Eribulin)的体内抑瘤活性，其中对照组包括DS-8201组和溶媒组(Vehicle)。
发明详述
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
在本文中，术语“HER2”和“HER2受体”可以互换使用，该蛋白也称为ErbB2、c-ERB2、c-ERB-2、NEU、HER-2/neu、p185(erbB2)或CD340。除非另外指明其来自非人物种，例如，“小鼠HER2”、“猴HER2”等，本文所表述的“HER2”是指人HER2的任何天然形式，可以具有如SEQ ID NO:234所示的氨基酸序列和/或如NCBI登记号NP_004439.2所示的全长HER2氨基酸序列，也可以由细胞(包括肿瘤细胞)天然地表达，或以HER2基因或cDNA转染的细胞表达。该术语包括天然存在的HER2等位基因变体和剪接变体、同种型、同源物和物种同源物。HER2可以从人体中分离，或可以通过重组或合成的方法产生。
HER2的胞外结构域由4个亚结构域组成，即亚结构域1(Subdomain 1，简称D1，约第1-195位氨基酸残基)、亚结构域2(Subdomain 2，简称D2，约第196-319位氨基酸残基)、亚结构域3(Subdomain 3，简称D3，约第320-488位氨基酸残基)、和亚结构域4(Subdomain 4，简称D4，约第489-630位氨基酸残基)(残基编号无信号肽)；其中，D2和D4是负责受体二聚化的富含半胱氨酸的结构域(Garrett等，Mol Cell 2003，11:495-505；Cho等，Nature 2003，421:756-760；Franklin等，Cancer Cell 2004，5:317-328)。
在本文中，“表达HER2的细胞”可以是天然存在的细胞或细胞株(例如，肿瘤细胞)，也可以是通过将编码HER2的核酸导入到宿主细胞中重组产生的。
在本文中，“双特异性抗体”或“双特异抗体”旨在包括任何能够特异性地结合两个不同抗原表位的抗体或抗原结合片段，其包含两个独立的抗原结合结构域且各自具有独特的抗原结合特异性。例如，本文所述的“互补性双特异抗体”特指一类双特异性抗体，其第一抗原结合结构域和第二抗原结合结构域分别结合至同一抗原上的不同表位。
单特异性抗体是指只有一种结合特异性的抗体或抗原结合片段，即单特异性抗体的抗原结合结构域结合至某单一抗原的单一表位。在一些实施方案中，所述的单特异性抗体的实例包括本发明的抗HER2单特异性抗体。
在本文中，“抗原结合结构域”或“抗原结合区”或“表位结合结构域”或“抗原结合多肽”可互换使用，是指抗体或抗原结合片段或其衍生物上的特定区域，该区域直接涉及与靶抗原的特异性相互作用，例如通过结合、空间位阻、使稳定/使不稳定、空间分布等方式与靶抗原相互作用以至达到动态平衡。本发明中“抗原结合结构域”也指所述抗体或抗原结合片段或其衍生物上的特定区域，该区域与HER2上的特定表位相互作用，通过结合、空间位阻、使稳定/使不稳定、空间分布等方式使二者之间的结合达至动态平衡。
“抗体”是指大体上由一个免疫球蛋白基因或多个免疫球基因、或其片段编码的能够特异性识别并结合抗原的多肽或蛋白质。公认的免疫球蛋白基因包含κ、λ、α、γ、δ、ε和μ恒定区基因，以及无数的免疫球蛋白可变区基因。轻链被分类为κ或λ。重链分为γ、μ、α、δ或ε，其依次分别定义了免疫球蛋白类别(Class)或同种型(Isotype)IgG、IgM、IgA、IgD和IgE，其中的几类可进一步分为亚类(Subclass)，例如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2。典型的免疫球蛋白(例如抗体)结构单元为四聚体，每个四聚体由相同的两对多肽链构成，每对具有一条“轻”链(约25kD)和一条“重”链(约50-70kD)。每条链的N端结构域定义了主要负责抗原识别的由约100至110个或更多氨基酸组成的可变(V)区。抗体重链由重链可变区(VH)和重链恒定区(CH)组成，其中重链恒定区通常包括三个域，CH1、CH2和CH3。轻链由轻链可变区(VL)和轻链恒定域(CL)组成，其中轻链恒定域通常包含一个域，CL。VH和VL的配对一起形成单个抗原结合位点。内源性VL由基因区段V(可变的)和J(接合的)编码，并且内源性VH由V、D(多样性)和J编码。VL或VH都包括高变区(Region of Hypervariability)或称为互补决定区(Complementarity Determining Region，简称CDR)和框架区(Framework Region，简称FR)。术语“可变区”或“V区”可互换地使用，是指从氨基端到羧基端以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4顺序排列的重链可变区或轻链可变区。术语“J区”是指编码包含CDR3和FR4的C-末端部分的可变区的子序列。V区或J区可以是天然存在的、重组的或者合成的。在本文中，抗体轻链可变区和/或抗体重链可变区有时可统称为“抗体可变区”，抗体轻链和/或抗体重链统称为“抗体链”。在某些实施方案中，本文提供的抗体或其抗原结合片段的FR可以与人种系序列相同，也可以是天然的或人工修饰的。
CDR和FR的位置可以使用本领域熟知的多种定义方法确定，例如，Kabat、Chothia、IMGT和Contact(参见：Kabat等,Sequences of Proteins of Immunological Interest,1991第五版,NIH公开号91-3242；Johnson等,Nucleic Acids Res 2001,29:205-206；Chothia&Lesk,J Mol Biol 1987,196:901-917；Chothia等,Nature 1989,342:877-883；Chothia等,J Mol Biol 1992,227:799-817；Al-Lazikani等,J Mol Biol 1997,273:927-748；Lefranc等,Nucleic Acids Research 1999,27:209–212；MacCallum等,J Mol Biol 1996,262:732-745)。抗原结合位点的定义还在以下文献中描述：Ruiz等,Nucleic Acids Res 2000,28:219-221；Lefranc,Nucleic Acids Res 2001,29:207-209；Lefranc,The Immunologist 1999,7:132-136；Lefranc等,Dev Comp Immunol 2003,27:55-77； MacCallum等,J Mol Biol 1996,262:732-745；Martin等,Proc Natl Acad Sci USA 1989,86:9268-9272；Martin等,Methods Enzymol 1991,203:121-153；Sternberg编辑,Protein Structure Prediction:A Practical Approach,1996 Oxford University Press,141-172。本发明包含任何一种定义方法来确定本发明的抗HER2互补性双特异抗体或本发明的抗HER2抗体或其抗原结合片段中的CDR，表1显示了使用不同定义方法所确定的抗体CDR氨基酸序列的位置编号。涵盖特定CDR的确切氨基酸残基数量随CDR的序列而变化。在明确了抗体可变区氨基酸序列的情况下，本领域技术人员能够通过包括不限于所述定义的常规方法确定所述抗体的CDR。
表1.不同定义方法确定的CDR1

1:表1中所有CDR定义的编号均依照Kabat等人提出的编号系统(Kabat等,Sequences of Proteins of Immunological Interest,
1991第五版,NIH公开号91-3242)。
此外，Kabat等人还定义了针对可变区序列的编号系统，其可应用于任何抗体。本领域的普通技术人员能够明确地将此“Kabat编号”系统应用到任何抗体的可变区序列，而无需依赖于抗体序列本身以外的任何实验数据来确定可变区序列。除非另外说明，本发明所述的抗HER2抗体、抗HER2互补性双特异抗体、或互补性双特异ADC的抗原结合结构域的可变区中特定氨基酸残基位置的编号均按照Kabat编号系统确定。
抗体以完整的免疫球蛋白形式存在，或者以通过多种肽酶消化产生的许多片段形式存在。尽管按照完整抗体的消化，定义了多种抗体片段，但是技术人员应当理解，可以通过化学裂解方法或者通过使用重组DNA方法合成该类片段。在本文中，术语抗体的“抗原结合片段”(或简称为“抗体部分”或“抗体片段”)是指含有一个或多个CDR的抗体部分或者其他任何能够与抗原(例如，HER2或HER2的胞外结构域)结合但不具有完整抗体结构的抗体片段。抗原结合片段可以与完整抗体一样具有特异性结合抗原的活性。优选的抗原结合片段还保留了能够内化进入表达靶抗原的细胞的能力。在某些实施方案中，抗原结合片段可以含有来自某特定人抗体的一个或多个CDR，移接至来自一个或多个不同人抗体的框架区。抗原结合片段包括但不限于：(ⅰ)“Fab”片段，由VH、VL、CL和CH1结构域组成的单价抗体片段；(ⅱ)“F(ab')2”片段，包含两个由铰链区的二硫键连接的Fab片段的二价片段；(ⅲ)“Fv”片段，由抗体单臂的VL和VH结构域组成，是含有完整抗原结合位点的最小抗体片段；(ⅳ)“Fd”片段，由VH和CH1结构域组成；(ⅴ)“单链Fv抗体(scFv)”或“单链抗体”，是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程化抗体(Huston等,Proc Natl Acad Sci USA 1988,85:5879-5883；Bird等,Science 1988,242:423-426)，该类scFv抗体在与细胞表面抗原结合时可以内化至细胞内(He等,J Nucl Med 2010,51:427-432；Fitting等,MAbs 2015,7:390-402)；此外，单链抗体还包括包含一对串联的Fv区段(VH-CH1-VH-CH1)的“线性抗体”，其与互补轻链多肽一起形成一对抗原结合区(Zapata等,Protein Eng1995,8:1057-1062；US5641870)；(ⅵ)“dAb”片段(Ward等,Nature 1989,341:544-546；WO90/05144A1)包含单个可变域，例如VH结构域。单结构域抗体(sdAb)是独立免疫球蛋白结构域；(ⅶ)“双功能抗体(Diabody)”为二价双特异性抗体，其中VH和VL结构域在单个多肽链上表达，但是使用的连接子太短而不能使同一链上的两个结构域配对，因此迫使这两个结构域与另一条链的互补结构域配对，形成两个抗原结合位点(Holliger等,Proc Natl Acad Sci USA 1993,90:6444-6448；Poljak等,Structure 1994,2:1121-1123；EP404097；WO93/11161)。
在本文中，术语“Fc区”或“Fc结构域”是指免疫球蛋白重链的C末端区域，其至少含有恒定区的一部分，例如除了第一恒定区(CH1)之外的免疫球蛋白重链恒定区。对于IgG，所述Fc区可以包含免疫球蛋白结构域CH2和CH3以及CH1和CH2之间的铰链区。本文所使用的Fc区包括天然序列Fc区和/或Fc区变体，并且可以是本发明的抗HER2抗体、抗HER2互补性双特异抗体或其ADC的一部分。在本领域中应理解，Fc区的边界可以变化，然而，人IgG重链Fc区通常被定义为在其氨基端包含第226位半胱氨酸残基或第230位脯氨酸残基，其根据EU编号系统/方案，如见于Kabat等,Sequences of Proteins of Immunological Interest,1991第五版,NIH公开号91-3242。
术语“抗HER2抗体”或“特异性结合HER2的抗体”是指特异性结合HER2的任何形式的抗体或其片段，且涵盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体和生物功能抗体片段，只要所述片段特异 性结合HER2即可。
在本文中，术语“特异性结合”或“结合特异性”或“对…具有特异性”或“结合”是指结合反应，该结合反应决定了在蛋白质的异质群体以及其它生物制品(例如，生物样品，如血液、血清、血浆或组织样品)中靶分子(例如，抗原)的存在，也就是说此结合对于靶分子是具有选择性的，并且能区分开那些不期望的或非特异性的相互作用。例如，与靶分子(可以是抗原)特异性结合的抗体是相较于该抗体与其它非靶分子的结合，该抗体结合该靶分子时具有更高的亲和力、更强的结合活性、更容易结合和/或结合持续时间更长。因此，根据需要，这种选择可以通过去除与其它ErbB/HER家族成员交叉结合的抗体来实现。可以将多种免疫测定方法用于选择与特定蛋白特异性地反应的抗体，例如ELISA测定法。通常在设定的测定条件下，抗体或结合剂与抗原的特异性或选择性结合反应会产生至少两倍于背景的信号，更一般地至少是10-100倍于背景的信号，并且基本上不以显著量与样品中存在的其它抗原结合。在某些实施例中，抗体与靶抗原特异性结合的平衡解离常数(KD)为＜1μΜ、＜100nM、＜10nM、＜1nM、或＜0.1nM。
在本文中，术语“单克隆抗体”是指从基本上具有同质性的抗体群中所获得的抗体，即，除了可能以少量存在的天然突变之外，构成抗体群中的各个抗体是相同的。单克隆抗体表现出对特定表位的高度结合特异性和亲和力。单克隆抗体可以通过Kohler等,Nature 1975,256:495最先描述的杂交瘤方法制备，也可以通过重组DNA方法制备(参见US4816567)，还可以从噬菌体抗体库，例如参照Clackson等,Nature 1991,352:624-628；Marks等,J Mol Biol 1991,222:581-597中描述的技术进行分离。
在本文中，术语“嵌合抗体”是指含有源自两种不同抗体的序列的抗体(例如，US4816567)，所述抗体通常源自不同物种。例如，嵌合抗体包含人类和啮齿类动物抗体片段，通常是人类恒定区和小鼠可变区。用于产生嵌合抗体的方法包含本领域一般技术人员已知的重组DNA和基因转染技术(例如Morrison等,Proc Natl Acad Sci USA 1984,81:6851-6855；US 5202238和US 5204244)。
在本文中，术语“人源化抗体或抗原结合片段”，是指包括来源于非人类动物的CDR、来源于人的FR区，以及来源于人的恒定区的抗体或抗原结合片段。人源化抗体任选的还包含至少一部分的人类免疫球蛋白的恒定区。由于人源化的抗体或抗原结合片段具有降低了的免疫原性，可以作为施用于人体的治疗剂。在一些实施方案中，所述非人类动物是哺乳类动物例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。在一些实施方案中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，抗体的其它部分基本上全部由人源序列组成。在一些实施方案中，人源化抗体可以通过以非人类物种抗体中的残基取代人免疫球蛋白FR框架区中相应的残基，以进一步修饰、改善和优化抗体的特异性、亲和力和/或活性。在一些实施方案中，所述来源于人的FR区可以包括与其来自的人源抗体相同的氨基酸序列，或其可以包括一些氨基酸改变，例如，不超过5、4、3、2、或1个氨基酸改变。在一些实施方式中，该氨基酸改变可以仅存在于重链FR区、仅存在于轻链FR区或同时存在于两条链中。
在本文中，术语“相应的人种系(Germline)序列”是指抗体可变区氨基酸序列或子序列，与所有其他已知人种系免疫球蛋白可变区氨基酸序列相比对，该抗体可变区氨基酸序列或子序列与参照的人种系免疫球蛋白可变区氨基酸序列具有最高的氨基酸序列同一性。相应的人种系序列可以是单独的框架区、单独的互补性决定区、框架区和互补性决定区、可变区、或包含可变区序列或子序列的其它组合。可以使用本文所述的方法，例如，使用BLAST、ALIGN或本领域已知的其它比对算法比对两个序列，来确定序列同一性。相应的人种系氨基酸序列可以与参照的人种系免疫球蛋白可变区氨基酸序列具有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％序列同一性。
本发明的抗HER2抗体、抗HER2互补性双特异抗体或ADC的抗体部分可以选自以下任一个或多个形式，包括嵌合形式、非人形式、人源化形式或全人形式，只要所述形式能够特异性地结合HER2即可。
如本文所用的术语“表位(Epitope)”是指蛋白质决定簇(Protein Determinant)，能够被抗体识别且特异性结合的抗原部分。表位通常由分子的表面基团如氨基酸或糖侧链组成，且通常具有特定的三维结构特征，以及特定的电荷特征。抗体或其抗原结合片段识别该表位的部分被称为互补位(Paratope)。
竞争性结合(Competitive Binding)和表位分组(Epitope Binning)也可以用于测定不同抗体是否结合于相同表位或重叠表位，例如使用Harlow和Lane编辑，Antibodies,A Laboratory Manual,Cold Spring Harbor Laboratory中描述的方法来评估。在一些实施方案中，通过竞争结合ELISA方法检测抗体或其抗原结合片段(例如包含选自表2和表3中鉴别的CDR和/或可变区的抗体或抗原结合片段)与其它抗体或其抗原结合片段之间彼此抑制与靶抗原(例如HER2)结合的活性，当结合活性减少至少约50％(例如50％、60％、70％、80％、90％、95％、99％或更多，或所列举数值之间的任何百分比)时，确定二者为竞争性结合，即结合于相同表位或重叠表位。在一些实施方案中，竞争性结合可以归因于共享的(Shared)或类似(例如部分重叠)的表位，或归因于抗体或抗原结合片段结合在附近表位而导致的位阻效应(参见Morris编辑,Methods in Molecular Biology 1998,第66卷,第55-66页)。在一些实施方案中，竞争性结合可以用来将结合于共用类似表位的抗体或抗原结合片段进行分组，例如竞争结合的抗体或抗原结合片段可以“分组”为具有重叠或接近表位的一组抗体或抗原结合片段，而不竞争的抗体或抗原结合片段则归为不具有重叠或 接近表位的另一组抗体或抗原结合片段。
术语“亲和力”或“结合亲和力”是指分子(例如受体或抗原)与其配对体(例如配体或抗体)之间相互作用的内在结合能力，即全部非共价相互作用总和的强度。除非另外陈述，本文中使用的“结合亲和力”是用于反映结合对的成员(例如受体与配体或抗原与抗体)之间一对一相互作用的内在结合能力。分子X对其配对体Y的亲和力通常可以用平衡解离常数(KD)表示，平衡解离常数是解离速率常数(kdis或koff)和结合速率常数(ka或kon)的比值。亲和力可以通过本领域已知的常见方法来测量，包括本发明中所使用的方法。
在本文中所使用的“内化(Internalization)或内吞(Endocytosis)”是指抗体或抗原结合片段、或双特异性抗体、或ADC与细胞表面靶抗原结合时，能够通过细胞脂质双层膜的内化(即胞吞作用)进入到细胞的内体(Endosome)，优选能够进入细胞中溶酶体(Lysosome)内的抗体或抗原结合片段、或双特异性抗体。在一些实施方案中，本发明所述的抗HER2互补性双特异抗体或其ADC结合于肿瘤细胞膜上的HER2后能够被内吞进入细胞，并能下调肿瘤细胞表面HER2的表达量。
在本文中，术语“抗体变异体”或“抗体变体”是指在原始抗体可变区中含有至少一个氨基酸突变的抗体多肽序列。变异体可以与未经修饰的抗体基本同源或基本相同。在一些实施方案中，在本发明的抗HER2抗体、抗HER2互补性双特异抗体或ADC的抗体部分的一个、两个、三个、四个、五个和/或六个CDR内具有氨基酸突变，以改善和优化所述抗体或抗原结合片段的性能，包括但不限于提高人源化程度、增强对HER2的结合亲和力或结合活性、提高内化效率、提高表达量、和/或提高稳定性(例如，降低或消除天冬氨酸异构化和/或天冬酰胺脱酰基的风险)。在一些实施方案中，在本发明的抗HER2抗体、抗HER2互补性双特异抗体或ADC抗体部分的一个、两个、三个和/或四个FR内具有氨基酸突变，以改善和优化所述抗体或抗原结合部分的性能，包括但不限于提高所述抗体或抗原结合部分的人源化程度、增强对HER2的结合亲和力或结合活性、提高表达量、和/或提高稳定性(例如，降低或消除天冬氨酸异构化和/或天冬酰胺脱酰基的风险)。在一些实施方案中，在本发明的抗HER2抗体、抗HER2互补性双特异抗体或ADC的抗体部分的CDR和FR内进行一个或多个氨基酸突变，以提高所述抗体或抗原结合片段的人源化程度、增强对HER2的结合亲和力或结合活性、提高内化率、提高表达量、和/或提高稳定性(例如，降低或消除天冬氨酸异构化和/或天冬酰胺脱酰基的风险)。在一些实施方案中，所述氨基酸突变包括氨基酸取代、去除、插入或其任意组合。
其中，氨基酸取代包括保守氨基酸取代和非保守氨基酸取代，保守氨基酸取代涉及用同类中的另一个氨基酸(化学性质或功能相似)进行置换，非保守取代涉及用不同类的氨基酸(化学性质或功能不相似)进行置换。本领域一般技术人员可以基于所涉及残基的极性、电荷、溶解性、疏水性、亲水性和/或两亲性质的相似性进行保守或非保守氨基酸取代。例如，(ⅰ)非极性(疏水)氨基酸包括丙氨酸(Ala，A)、亮氨酸(Leu，L)、异亮氨酸(Ile，I)、缬氨酸(Val，V)、脯氨酸(Pro，P)、苯丙氨酸(Phe，F)、色氨酸(Trp，W)和蛋氨酸(Met，M)；(ⅱ)极性中性氨基酸包括甘氨酸(Gly，G)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、半胱氨酸(Cys，C)、酪氨酸(Tyr，Y)、天冬酰胺(Asn，N)和谷氨酰胺(Gln，Q)；(ⅲ)带正电荷的(碱性)氨基酸包括精氨酸(Arg，R)、赖氨酸(Lys，K)和组氨酸(His，H)；(ⅳ)带负电的(酸性)氨基酸包括天冬氨酸(Asp，D)和谷氨酸(Glu，E)。一般而言，保守氨基酸取代不会实质上改变蛋白质的功能性质，非保守氨基酸取代可能导致蛋白质的性质或功能发生很大变化。尽管对引入氨基酸序列突变的位点或区域可以预先确定，但非保守取代所带来的潜在的蛋白质性质或功能的改变却是无法预见的。
在本文中，对于两个或多个多肽序列，术语“相同的”或“同一性”或“同一性百分比”或“百分比序列同一性”可以互换使用，是指在进行氨基酸序列比对时，并在引入必要的间隔使相同氨基酸数目达到最多后，候选序列中与参比序列相同的氨基酸残基数占所述候选序列总氨基酸残基数的百分比率。所述氨基酸残基的保守替代可以认为或可以不认为是相同残基。可以通过本领域公开的工具，例如BLASTp、ClustalW2(也可参见Higgins等,Methods Enzymol 1996,266:383-402；Larkin等,Bioinformatics 2007,23:2947-2948)和ALIGN或Megalign(DNASTAR)软件，对序列进行比对以确定氨基酸序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法来进行序列比对。
在本文中，当涉及蛋白质时，术语“分离的”表示该蛋白基本上不含在天然状态下与其结合的其他细胞组分，优选处于同质状态，例如，分离的蛋白可以是从天然或自然环境中移出的。分离的蛋白可以是冻干的或者是水溶液。通常，其纯度和均一性可以使用分析化学技术(例如，聚丙烯酰胺凝胶电泳或高效液相色谱法)来确定。在分离的制备物中蛋白质是基本上纯化的。术语“纯化的”表示蛋白在非还原电泳凝胶中基本上只产生一条带。特别地，这意味着蛋白为至少85％纯度、更优选地至少95％纯度、以及最优选地至少99％纯度。就本发明而言，在一些实施方案中，在宿主细胞中表达的重组蛋白则被视为是分离的，同样也适用于通过任何本领域技术人员熟知的技术分开、分级(Fractionate)、或部分或基本纯化的天然 的或重组的蛋白。在一些实施方案中，“分离的抗体”指基本上不含具有不同抗原特异性的其它抗体的抗体(例如，特异性地结合HER2的分离的抗体基本上不含特异性地结合除HER2之外的抗原的抗体)。然而，特异性地结合HER2的分离的抗体可以与其它抗原(如来自其它物种的HER2蛋白，例如猴HER2)具有交叉结合反应性。而且，分离的抗体可以基本上不含其它细胞材料和/或化学物质。在一些实施方案中，包含在载体中的编码本发明多肽或蛋白(例如抗HER2抗体、抗HER2互补性双特异抗体)的重组多核苷酸被视为分离的。分离的多核苷酸的其它例子包括在异源宿主细胞中包含的重组多核苷酸或溶液中的纯化(部分或基本地)的多核苷酸。
在本文中，术语“工程化”包括对多肽或蛋白质主链的任意操作，或对天然存在的或重组的蛋白质或多肽的翻译后修饰。工程化包括对氨基酸序列进行突变、对糖基化或各氨基酸侧链基团的修饰，以及这些方法的组合。
如本文所用的，术语“多肽”是指氨基酸及其等同物的聚合物，而不是指产物的具体长度；因此，“肽”和“蛋白质”包含在多肽的定义内。多肽的定义内还包含本发明定义的“抗体”。
双重可变结构域(Dual Variable Domain,简称DVD)免疫球蛋白(DVD-Ig)是在正常IgG抗体的重链和轻链的N末端分别再接入另一个抗体的重链可变区(VH)和轻链可变区(VL)结构域，产生一个含有两种不同抗原结合结构域的四价分子。因此，DVD-Ig的每半侧含有一条重链多肽和一条轻链多肽，二聚化后构成两个独立的抗原结合结构域，其中一个是Fv片段，另一个是Fab片段或IgG结构域。DVD-Ig可以是双特异性的，即能够结合一种抗原的两个不同表位，或者结合两种不同抗原。
在本文中，术语“表达载体”或“载体”是指可将编码某蛋白的多核苷酸或核酸操作性地插入其中并使该蛋白获得表达的一种运载工具。载体可用于转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。
在本文中，术语“宿主细胞”是指导入外源多核苷酸或核酸和/或载体的细胞。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，并且可能包含突变。本发明中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
在本文中，术语“受试者”、“患者”或“个体”可以互换使用，包括但不限于：哺乳动物，包含例如人、非人灵长类动物(例如，猴)、小鼠、猪、狗、猫、牛、山羊、兔、大鼠、豚鼠、仓鼠、马、绵羊或其它非人类哺乳动物；非哺乳动物，包含例如非哺乳类脊椎动物，如鸟(例如，鸡、鸸鹋或鸭)或鱼；以及非哺乳类无脊椎动物。在一些实施例中，本发明的用途或方法所涉及的受试者和药物组合物用于(预防性地和/或治疗性地)治疗非人类动物。
在本文中，对某种疾病或症状的“治疗(Treating；Treatment；或To treat)”是指减轻(Alleviating或To alleviate)某种疾病或症状，降低某种疾病或症状发作或发展的速度，减少发展出某种疾病或症状的风险，或延迟与某种疾病或症状相关的症状发展，减少或终止与某种疾病或症状相关的症状，产生某种疾病或症状的完全或部分的逆转，治愈某种疾病或症状，或以上的组合。
术语“治疗有效量”或“有效剂量”是指以需要的剂量并持续需要的时间段，有效实现预防或改善与疾病或病症有关的症状和/或减轻疾病或病症严重程度的剂量或浓度。本发明的制剂、抗体或其抗原结合片段、ADC、或组合物的治疗有效量可以根据多种因素而变动，例如疾病状态、个体的年龄、性别和重量、以及抗体或抗体部分或ADC在个体中激发所需反应的能力。治疗有效量也可被认为是制剂、抗体或其抗原结合片段、ADC、或组合物的任何有毒或有害作用不及治疗有益作用。术语“有效量”是指足以对受试者提供临床上的益处(包括但不限于改善、缓解或减轻疾病、病症或其相关症状，延迟或停止疾病进展)的活性成分或药剂的量。
在本文中，“低水平HER2”、“低表达HER2”、“低HER2表达”、“HER2低表达”、或“HER2低水平表达”是指肿瘤活检的石蜡包埋组织切片经免疫组织化学法(Immunohistochemistry Staining，简称IHC)测定(例如)时ErbB2/HER2蛋白质染色强度得分为IHC 1+(在多于10％的肿瘤细胞中检测到模糊的/几乎不能看出的膜染色，仅在其一部分膜中细胞被染色)，或者当得分为IHC 2+(在多于10％的肿瘤细胞中观察到弱至中等的全膜染色)时，通过荧光原位杂交法(Fluorescence in Situ Hybridization，简称FISH)测定(例如InformTM[Ventana]，或PathVysionTM[Vysis])进一步确定肿瘤细胞中HER2基因扩增程度的得分为阴性(IHC 2+/FISH-)的癌细胞/肿瘤、受试者或患者。大多数的HER2低表达肿瘤的HER2表达水平是显著高于正常组织的。正常组织的HER2表达水平可以根据本领域技术人员可用的任何方法来确定。
“HER2阴性”或“HER2表达阴性”或“表达有限水平HER2”是指如上所述的HER2低表达和IHC测定得分为0(未观察到染色或膜染色存在于不到10％的肿瘤细胞中)的癌细胞/肿瘤、受试者或患者。
“HER2阳性”或“HER2表达阳性”或“HER2过表达”是指IHC测定得分为IHC 3+(在超过10％的肿瘤细胞中观察到强烈的完整膜染色)的癌细胞/肿瘤、受试者或患者。HER2阳性还包括IHC测定得分 为IHC 2+，同时FISH测试(例如，减法探针技术显色原位杂交[SPoT-Light HER2 CISH]测试、Inform双原位杂交[Inform HER2Dual ISH]测试)中评分为阳性的癌细胞/肿瘤、受试者或患者(IHC 2+/FISH+)。
在本文中，ADC的“旁观者效应(Bystander Effect)”或“旁观者杀伤效应(Bystander Killing Effect)”是由游离的具有膜通透性的小分子毒素(例如，抗癌剂)介导的，该小分子毒素由靶标分子阳性(如HER2阳性)癌细胞释放并被动扩散到肿瘤微环境中，进而杀死邻近的细胞，包括不表达或低表达靶标分子(如HER2阴性)而对ADC不敏感的邻近癌细胞。
在本文中，术语“药学上可接受的”或“药用可接受的”是指所指的载剂、溶媒、稀释剂、辅料和/或盐，总的来说在化学上和/或在物理上与制剂中的其他配料相兼容，并在生理上与受试者相兼容。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，或在一个实施方案中为小10％的下限和大10％的上限，或在另一个实施方案中为小15％的下限和大15％的上限，或在另一个实施方案中为小20％的下限和大20％的上限。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
如本文中所用，术语“包含”或“包括”或“含有”或“具有”或“涉及”可互换使用，意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”或“含有”或“具有”或“涉及”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。
如本文中所用，术语“任选的”表示其修饰的对象存在或者不存在，例如“药盒包含任选的至少一种另外的肿瘤治疗剂”表示该药盒可包含或不包含至少一种另外的肿瘤治疗剂。
如本文中所提及的“一些实施方案”、“一个实施方案”、“一个具体实施方案”、或“具体实施方案”或其组合表示所描述的与所述实施方案相关的特定特征、结构或特性包含于本发明的至少一个实施方案中。因此，在本说明书全文各处出现前述用语未必都指同一个实施方案。此外，所述特定特征、结构或特性可在一个或多个实施方案中以任意适宜方式组合。
除非上下文另有明确指示，单数术语涵盖复数的指示对象，反之亦然。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本发明的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于本发明申请者可获得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。
在以下章节中将更详细地描述本发明的各个方面。
1、抗HER2抗体或其抗原结合片段
一方面，本发明提供了一种分离的抗HER2抗体或其抗原结合片段，所述抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外结构域。
本发明的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区的D1、D3和/或D4，优选HER2胞外区的D1或D3。本发明的抗HER2互补性双特异抗体可以利用两个单特异性抗HER2抗体的抗原结合结构域来构建，所述的两个单特异性抗体能够各自结合于HER2胞外区的不同表位，并且在抗原竞争性结合测试中所述的两个单特异性抗体不存在竞争抑制关系(即抗原结合表位不重叠)。
在一些实施方案中，可以利用两个单特异性抗HER2抗体的抗原结合结构域来构建本发明的抗HER2互补性双特异抗体，所述的两个单特异性抗体能够同时结合于HER2胞外区中的不同表位，进一步，所述的两个单特异性抗体能够同时结合至HER2胞外区的D1、D3和D4中的任意两个表位，优选地，可同时结合至HER2胞外区的D1和D3，例如，其中，第一抗HER2抗体的抗原结合结构域结合HER2胞外区的D3，第二抗HER2抗体的抗原结合结构域结合HER2胞外区的D1。
所述抗HER2抗体或其抗原结合片段对表达HER2的肿瘤细胞具有较强的结合活性，所述表达HER2的肿瘤细胞包括HER2高表达肿瘤细胞(例如，乳腺癌细胞SKBR-3、乳腺导管癌细胞BT474、胃癌细胞NCI-N87以及卵巢癌细胞SKOV-3)、表达中度水平HER2的肿瘤细胞(例如，乳腺癌细胞JIMT-1)、HER2低表达肿瘤细胞(例如，人膀胱癌细胞RT-112、乳腺癌细胞ZR-75-1和T47D)和/或表达有限水平HER2的肿瘤细胞(例如，乳腺癌细胞MCF-7)。所述抗HER2抗体或其抗原结合片段与HER2的结合亲和力(KD)值＜5×10-8M，优选的是＜1×10-8M，＜5×10-9M，或＜1×10-9M，更优选的是＜1×10-9M。所述抗HER2抗体或其抗原结合片段不与ErbB/HER家族其它成员(包括EGFR、HER3和HER4)发生交叉结合反应。
所述抗HER2抗体或其抗原结合片段不影响表达于细胞表面的HER2及其下游信号转导通路的调控。在一些实施方案中，所述抗HER2抗体或其抗原结合片段不诱导、不阻断、不抑制HER2胞内结构域的酪氨酸残基磷酸化和/或去磷酸化，所述HER2胞内结构域的酪氨酸残基磷酸化位点包括但不限于Y877、Y1221/1222和Y1248。在一些实施方案中，所述抗HER2抗体或其抗原结合片段不诱导、不阻断、不抑制依赖于或不依赖于配体诱导的HER2二聚化(包括HER2:HER4二聚化和/或HER2:HER3二聚化)及其下 游的信号转导通路，所述配体包括NRG-1或Heregulins。
本发明的抗HER2抗体还可以任选地包括F(ab’)2、Fab、Fab’、Fv、scFv、scFv-Fc、单结构域抗体(sdAb)，或具有IgG类型。本发明的抗体可以是鼠源抗体、嵌合抗体、人源化抗体、全人抗体，可以是单克隆抗体、多克隆抗体、单特异性抗体、双特异性抗体、多特异性抗体或抗体片段，只要所述抗体能够特异性地识别HER2胞外区的D1、D3和/或D4(优选D1和/或D3)的表位，且对表达于肿瘤细胞表面的HER2及其信号转导通路不具有影响作用即可。在一些实施方案中，所述抗HER2抗体选自小鼠抗人HER2抗体及其人源化抗体和优化抗体。在一些实施方案中，所述抗HER2抗体包括scFv、scFv-Fc、Fab片段和/或具有IgG类型，例如，特异性结合HER2胞外区的D3的抗体可采用scFv或scFv-Fc形式，特异性结合HER2胞外区的D1的抗体可采用Fab或IgG形式，其中Fab和scFv可以随意转换，转换方法在本领域是已知的(参见，例如，Zhou等,Mol Cancer Ther 2012,11:1167-1476所描述的方法)。
另一方面，本发明提供包含表2中所示抗体的CDRs和/或可变区的抗HER2抗体或其抗原结合片段，其识别HER2胞外区的D3，并且不诱导、不阻断、不抑制HER2胞内结构域的酪氨酸残基磷酸化和/或去磷酸化，和/或不诱导、不阻断、不抑制依赖于或不依赖于NRG-1诱导的HER2二聚化及其下游信号转导通路。本发明还提供能够识别HER2胞外区D1的抗HER2抗体或其抗原结合片段，其不诱导、不阻断、不抑制HER2胞内结构域的酪氨酸残基磷酸化和/或去磷酸化，和/或不诱导、不阻断、不抑制依赖于或不依赖于NRG-1诱导的HER2二聚化及其下游信号转导通路，优选地，所述抗体包含如表3所示的抗体的CDRs和/或可变区的抗HER2抗体或其抗原结合片段。本发明的抗HER2抗体或其抗原结合片段，还包括识别HER2胞外区D4的抗HER2抗体或其抗原结合片段的CDRs、可变区、或轻链和重链。
本发明的抗HER2抗体的重链可变区CDRs和轻链可变区CDRs由Kabat编号系统定义。然而，如本领域所公知的，CDR区也可以基于重链/轻链可变区序列的其它诸如Chothia和IMGT、AbM或Contact编号系统/方法定义，以其它编号系统/方法定义的CDR区与本发明采用的Kabat所定义的CDR区均在本发明保护范围之内。
表2.抗体mAb2117及其人源化抗体的CDR区以及重链可变区和轻链可变区的氨基酸序列ID号

表3.抗体mAb2126及其人源化抗体的CDR区以及重链可变区和轻链可变区的氨基酸序列ID号

在一些实施方案中，本发明的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区的D3，其包含如SEQ ID NO:1所示的VH氨基酸序列和/或如SEQ ID NO:2所示的VL氨基酸序列。
在一些实施方案中，本发明的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区的D1，其包含如SEQ ID NO:3、7、或9所示的VH氨基酸序列和/或如SEQ ID NO:4、8、或10所示的VL氨基酸序列。在一个实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含如SEQ ID NO:3所示的VH氨基酸序列和/或如SEQ ID NO:4所示的VL氨基酸序列。在一个实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含如SEQ ID NO:7所示的VH氨基酸序列和/或如SEQ ID NO:8所示的VL氨基酸序列。在一个实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含如SEQ ID NO:9所示的VH氨基酸序列和/或如SEQ ID NO:10所示的VL氨基酸序列。
在一些实施方案中，本发明的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区的D4，其包含如SEQ ID NO:5所示的VH氨基酸序列和/或如SEQ ID NO:6所示的VL氨基酸序列。
在一些实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2抗体或其抗原结合片段包含SEQ ID NO:1所示的VH氨基酸序列中的一个或多个CDR或如SEQ ID NOs:11、14和32所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2抗体或其抗原结合片段进一步包含SEQ ID NO:2所示的VL氨基酸序列中的一个或多个CDR或如SEQ ID NOs:38、46和49所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，所述抗HER2抗体或其抗原结合片段包含如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:14所示的HCDR2和如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3。
在一些实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含SEQ ID NO:3所示的VH氨基酸序列中的一个或多个CDR或如SEQ ID NOs:98、100和110所示的氨基酸序列或其变异体，SEQ ID NO:7所示的VH氨基酸序列中的一个或多个CDR或如SEQ ID NOs:205、206和207所示的氨基酸序列或其变异体，和/或SEQ ID NO:9所示的VH氨基酸序列中的一个或多个CDR或如SEQ ID NOs:211、212和213所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含SEQ ID NO:4所示的VL氨基酸序列中的一个或多个CDR或如SEQ ID NOs:114、116和139所示的氨基酸序列或其变异体，SEQ ID NO:8所示的VL氨基酸序列中的一个或多个CDR或如SEQ ID NOs:208、209和210所示的氨基酸序列或其变异体，和/或SEQ ID NO:10所示的VL氨基酸序列中的一个或多个CDR或如SEQ ID NOs:214、215和216所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段包含：如SEQ ID NO:98所示的HCDR1、如SEQ ID NO:100所示的HCDR2和如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:114所示的LCDR1、如SEQ ID NO:116所示的LCDR2和如SEQ ID NO:139所示的LCDR3；或者，如SEQ ID NO:205所示的HCDR1、如SEQ ID NO:206所示的HCDR2和如SEQ ID NO:207所示的HCDR3，以及如SEQ ID NO:208所示的LCDR1、如SEQ ID NO:209所示的LCDR2和如SEQ ID NO:210所示的LCDR3；或者，如SEQ ID NO:211所示的HCDR1、如SEQ ID NO:212所示的HCDR2和如SEQ ID NO:213所示的HCDR3，以及如SEQ ID NO:214所示的LCDR1、如SEQ ID NO:215所示的LCDR2和如SEQ ID NO:216所示的LCDR3。
在一些实施方案中，本发明的特异性结合HER2胞外区的D4的抗HER2抗体或其抗原结合片段包含SEQ ID NO:5所示的VH氨基酸序列中的一个或多个CDR或如SEQ ID NOs:199、200和201所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，本发明的特异性结合HER2胞外区的D4的抗HER2抗体或其抗原结合片段包含SEQ ID NO:6所示的VL氨基酸序列中的一个或多个CDR或如SEQ ID NOs:202、203和204所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或本发明所述的任何其它变异体。
在一些实施方案中，本发明的特异性结合HER2胞外区的D4的抗HER2抗体或其抗原结合片段包含如SEQ ID NO:199所示的HCDR1、如SEQ ID NO:200所示的HCDR2和如SEQ ID NO:201所示的HCDR3，以及如SEQ ID NO:202所示的LCDR1、如SEQ ID NO:203所示的LCDR2和如SEQ ID NO:204所示的LCDR3。
在另一方面，可以使用本发明的抗HER2抗体或其抗原结合片段的VH和/或VL作为起始材料进行工程化改造，以制备成更适合施用于人体的本文所述的抗体。可以通过对一个或两个可变区(即，VH和/或VL)内的一个或多个氨基酸残基进行突变来对抗体进行工程化改造，例如，对一个或多个CDR区，和/或一个或多个FR框架区内的一个或多个氨基酸残基进行突变。
在一些实施方案中，通过CDR移植(Grafting)来对本发明的抗HER2抗体可变区进行工程化改造。抗体主要通过重链可变区和轻链可变区的六个CDR的氨基酸残基与靶抗原相互作用。因此，各个抗体之间的CDR区内的氨基酸序列比CDR区之外的序列(例如FR)更加多样化。因为CDR区的氨基酸序列负责抗体与抗原大多数的相互作用，所以可以通过构建表达载体来表达重组抗体以模拟特定的天然存在抗体的特性，所述表达载体包含将来自特定的天然存在抗体的CDR序列移植到另一个具有不同特性抗体的FR序列(参见，例如，Riechmann等,Nature 1998,332:323-327；Jones等,Nature 1986,321:522-525；Queen等,Proc Natl Acad Sci USA 1989,86:10029-10033；还可参见US5225539、US5530101、US5585089、US5693762、和US6180370)。
本发明的抗HER2抗体还可以包含不同的框架区序列。此类框架区序列可以从DNA公共数据库或已公开的涉及种系抗体基因序列的参考文献中获得。例如，人重链和轻链可变区基因的种系DNA序列可见 于“V Base”人种系序列数据库中(可从网址www.mrc-cpe.cam.ac.uk/vbase处获得)；以及Kabat等,Sequences of Proteins of Immunological Interest,1991第五版,NIH公开号91-3242；Tomlinson等,J Mol Biol 1992,227:776-798；及Cox等,Eur J Immunol 1994,24:827-836；以引用的方式将其各自的内容明确地并入本文。作为另一个实例，人重链和轻链可变区基因的种系DNA序列可见于IMGT数据库中，例如在人类免疫球蛋白中发现的以下的重链种系序列能够通过IMGT登记号获得：IGHV3-23(DP47；VH26；V3-23)或IGHV7-4。作为另一个实例，在人免疫球蛋白中发现的以下的轻链种系序列能够通过IMGT登记号获得：IGKV1-39或IGKV1-39*01。
可以使用本领域技术人员公知的称为Gapped BLAST的序列相似性搜索方法中的一种，基于经编译的蛋白序列数据库对抗体氨基酸序列进行比较(Altschul等,Nucleic Acids Res 1997,25:3389-3402)。
用于本发明的抗HER2抗体中优选的框架序列是与本发明鼠源母本抗体框架序列在结构上相似的(或同源性高的)受体框架区。在一些实施方案中，可以将VH或VL的CDR1区序列、CDR2区序列和CDR3区序列分别移植到受体框架区，所述的受体框架区具有与其所在种系免疫球蛋白基因相同或同源性最高的序列。在一个具体实施方案中，本发明选择将SEQ ID NO:1或3所示的VH和SEQ ID NO:2或4所示的VL的CDR区分别移植到人源IgG FR区得到人源化抗体，所述人源化抗体不仅能够保持与包含SEQ ID NO:1所示的VH和SEQ ID NO:2所示的VL氨基酸序列，或者与包含SEQ ID NO:3所示的VH和SEQ ID NO:4所示的VL氨基酸序列的母本抗体相似的抗原结合活性，而且不会影响HER2下游信号传导通路，例如，不影响HER2胞内结构域的酪氨酸残基的磷酸化(如，Y1248磷酸化)和/或配体(如，NRG-1或Heregulins)诱导的HER2二聚化及其介导的下游信号传导通路。
此外，还可以将结合至HER2的其它抗HER2抗体的VH和VL序列(或CDR序列，或全长重链和全长轻链序列)与本发明的抗HER2抗体的VH和VL序列(或CDR序列、或全长重链和全长轻链序列)进行“混合和匹配”。优选地，当将VH和VL链(或这些链内的CDR，或全长重链和全长轻链序列)进行混合和匹配时，来自特定的VH/VL对的VH序列被结构相似的VH序列进行替代。同样地，优选地，来自特定的VH/VL对的VL序列被结构相似的VL序列进行替代。同样地，应当用结构相似的全长重链序列替换来自具体全长重链/全长轻链对的全长重链序列。同样地，应当用结构相似的全长轻链序列替换来自具体全长重链/全长轻链对的全长轻链序列。
因此，在一个实施方案中，本发明的抗体或其抗原结合片段包括：(a)重链可变区，该重链可变区包含表2或表3所列出的氨基酸序列；以及(b)轻链可变区，该轻链可变区包含表2或表3所列出的氨基酸序列，或者，另一个抗HER2抗体的VL，其中，该抗体特异性地结合HER2胞外区的D3或D1。
在一个实施方案中，本发明的抗体或其抗原结合片段包括：(a)重链可变区，该重链可变区包含表2或表3所列出的氨基酸序列，或另一个抗HER2抗体的VH，其中，该抗体特异性地结合HER2胞外区的D3或D1；以及(b)轻链可变区，该轻链可变区包含表2或表3所列出的氨基酸序列。
在一个具体实施方案中，本发明的人源化抗体或其抗原结合片段包含与SEQ ID NO:52所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VH氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D3。
在一个具体实施方案中，本发明的人源化抗体或其抗原结合片段包含与SEQ ID NO:81所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VL氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D3。
在一个实施例中，本发明的人源化抗体包含与SEQ ID NO:52所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VH氨基酸序列，以及与SEQ ID NO:81所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VL氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D3。
在一个具体实施方案中，本发明的人源化抗体或其抗原结合片段包含与SEQ ID NO:143或170所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VH氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D1。
在一个具体实施方案中，本发明的人源化抗体或其抗原结合片段包含与SEQ ID NO:171所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VL氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D1。
在一个实施例中，本发明的人源化抗体包含与SEQ ID NO:143或170所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VH氨基酸序列，以及与SEQ ID NO:171所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的VL氨基酸序列。所述抗体或其抗原结合片段特异性地结合HER2胞外区的D1。
在一些实施方案中，可以将CDR序列移植到与种系序列相比包含一个或多个突变的框架区上，例如，使框架区内的氨基酸残基发生突变可以维持或增强抗体的抗原结合能力(参见，例如，US5530101、US5585089、US5693762、和US6180370)。在一些实施方案中，将本发明所述母本抗体的CDR移植到与种系序列相比具有一个或多个突变的受体框架区上，可以提高本发明的抗HER2抗体的人源化程度和/或改善抗原结合亲和力。
在一些具体实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2人源化抗体的FR区具有一个或多个氨基酸突变，以改善人源化抗体的抗原结合亲和力，例如，将SEQ ID NO:52所示的VH的FR区进行一个或多个氨基酸突变，进一步，可在HFR1进行氨基酸突变，例如，在HFR1第30位(对应于SEQ ID NO:52所示的VH氨基酸序列第30位)的丝氨酸残基进行氨基酸突变；和/或将SEQ ID NO:81所示的VL的FR区进行一个或多个氨基酸突变，进一步，可在LFR1、LFR2和/或LFR3进行一个或多个氨基酸突变，具体地，在LFR1第2位的天冬氨酸残基、第4位的蛋氨酸残基，LFR2第9位(对应于SEQ ID NO:81所示的VL氨基酸序列第43位)的丙氨酸残基，LFR3第29位(对应于SEQ ID NO:81所示的VL氨基酸序列第85位)的苏氨酸残基进行一个或多个氨基酸突变。
在一个具体实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2人源化抗体相对于SEQ ID NO:52所示的VH氨基酸序列在HFR区具有氨基酸突变S30N，和/或其相对于SEQ ID NO:81所示的VL氨基酸序列在LFR区具有一个或多个氨基酸突变，包括D2I、M4L、A43S、T85V，能够显著提高所述抗体的抗原结合亲和力。
在一些具体实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2人源化抗体的FR区具有一个或多个氨基酸突变，以提高本发明抗体的人源化程度，例如，将SEQ ID NO:52所示的VH的FR区进行一个或多个氨基酸突变，进一步，可在HFR1和/或HFR2进行氨基酸突变，例如，可在HFR1第29位的酪氨酸残基，和/或HFR2第14位(对应于SEQ ID NO:52所示氨基酸序列第49位)丙氨酸残基进行氨基酸突变；和/或将SEQ ID NO:81所示的VL的FR区进行一个或多个氨基酸突变，进一步，可在LFR3进行氨基酸突变，例如，在LFR3第31位(对应于SEQ ID NO:81所示的VL氨基酸序列第87位)的苯丙氨酸残基进行氨基酸突变，具体地，所述特异性结合HER2胞外区的D3的抗HER2人源化抗体相对于SEQ ID NO:52所示的VH氨基酸序列在HFR具有以下氨基酸突变Y29F和/或A49S，和/或其相对于SEQ ID NO:81所示的VL氨基酸序列在LFR区具有氨基酸突变F87Y，能够提高所述抗体的人源化程度。
在一些具体实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2人源化抗体的FR区具有一个或多个氨基酸突变，例如，将SEQ ID NO:143所示的VH的FR区进行一个或多个氨基酸突变，可以在HFR1和/或HFR3进行1个或多个氨基酸突变，以提高本发明抗HER2抗体的人源化程度。优选地，在HFR1第9位的丝氨酸残基、第18位的缬氨酸残基，和/或在HFR3第3位的缬氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第68位)、第4位的苯丙氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第69位)、第6位的亮氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第71位)、第10位的缬氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第75位)、第17位的异亮氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第82位)、第29位的苯丙氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第91位)进行一个或多个氨基酸突变。还可以将SEQ ID NO:171所示的VL的FR区进行一个或多个氨基酸突变，以提高本发明的抗HER2抗体的人源化程度，例如，可以在LFR2的第15位的天冬酰胺残基(对应于SEQ ID NO:171所示的VL氨基酸序列第49位)进行氨基酸突变。
在一个具体实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2人源化抗体相对于SEQ ID NO:143所示的VH氨基酸序列在HFR区具有以下一个或多个氨基酸突变：S9G、V18L、V68T或V68S、F69I、L71V或L71R、V75K或V75T、I82L或I82M、F91Y。在一些实施例中，特异性结合HER2胞外区的D1人源化抗体相对于SEQ ID NO:143所示的VH氨基酸序列在HFR区包含以下一个或多个氨基酸突变：V68T、F69I、L71R、V75K和F91Y，可显著提高所述抗体的人源化程度。
在一些实施方案中，本发明对人源化抗体的VH和/或VL的CDR区进行氨基酸突变，以改善抗体的一种或多种特性，例如，提高人源化程度、改善抗原结合亲和力、提高表达量、增强稳定性(如，降低潜在的天冬氨酸异构化和/或天冬酰胺脱酰胺风险)。可以进行定点突变或PCR诱导突变，并且能够以本领域已知的体外或体内检测方法来评价所述突变对抗体的功能特性的影响。突变可以是氨基酸取代、添加或缺失，优选氨基酸取代。特别地，重链CDR或轻链CDR区内不超过1个、2个、3个、4个、5个、6个、7个、8个、9个、或10个氨基酸残基发生突变。
在一些具体实施方案中，本发明的特异性结合HER2胞外区的D3的抗HER2人源化抗体的重链CDR区或轻链CDR区内具有不超过1个、2个、3个、4个、5个、6个、7个、8个、9个、或10个氨基酸突变。
在一个具体实施方案中，将SEQ ID NO:52所示的VH中的CDR区进行1个或多个氨基酸突变，进一步，可以在HCDR1、HCDR2和/或HCDR3进行1个、2个、3个、4个、或5个氨基酸突变。例如，可 以在HCDR1的第1位丝氨酸残基(对应于SEQ ID NO:52所示的VH第31位)，在HCDR2的第5位甘氨酸残基(对应于SEQ ID NO:52所示的VH第53位)、第7位丝氨酸残基(对应于SEQ ID NO:52所示的VH第55位)、第9位苏氨酸残基(对应于SEQ ID NO:52所示的VH第57位)、第12位脯氨酸残基(对应于SEQ ID NO:52所示的VH第60位)、第13位天冬氨酸残基(对应于SEQ ID NO:52所示的VH第61位)、第14位丝氨酸残基(对应于SEQ ID NO:52所示的VH第62位)，和/或在HCDR3的第3位丙氨酸残基(对应于SEQ ID NO:52所示的VH第97位)进行1个或多个氨基酸突变。
在一个实施例中，将SEQ ID NO:52所示的VH中的HCDR1、HCDR2和/或HCDR3进行1个或多个氨基酸突变，例如，对SEQ ID NO:52所示VH的HCDR2第7位丝氨酸残基(对应于SEQ ID NO:52所示的VH第55位)、第9位苏氨酸残基(对应于SEQ ID NO:52所示的VH第57位)、第12位脯氨酸残基(对应于SEQ ID NO:52所示的VH第60位)、第13位天冬氨酸残基(对应于SEQ ID NO:52所示的VH第61位)、第14位丝氨酸残基(对应于SEQ ID NO:52所示的VH第62位)，和/或对HCDR3的第3位丙氨酸残基(对应于SEQ ID NO:52所示的VH第97位)进行氨基酸突变。
在一个具体实施方案中，将SEQ ID NO:81所示的VL中的CDR区进行1个或多个氨基酸突变，进一步，可以在LCDR1、LCDR3和/或LCDR3进行1个、2个、或3个氨基酸突变。例如，可以在LCDR1第7位缬氨酸残基(对应于SEQ ID NO:81所示的VL第27c位)、第8位组氨酸残基(对应于SEQ ID NO:81所示的VL第27d位)、第11位甘氨酸残基(对应于SEQ ID NO:85所示的VL第29位)，在LCDR2第6位苯丙氨酸残基(对应于SEQ ID NO:81所示的VL第55位)，和/或在LCDR3第1位丝氨酸残基(对应于SEQ ID NO:81所示的VL第89位)、第8位酪氨酸残基(对应于SEQ ID NO:81所示的VL第96位)进行1个或多个氨基酸突变。
在一个实施例中，本发明对上述特异性结合HER2胞外区的D3的抗HER2人源化抗体的VH和VL的CDR区进行多个氨基酸突变，以提高本发明所述抗体的人源化程度、提高表达量、和/或提高稳定性(例如降低潜在的天冬氨酸异构化风险)。具体地，对SEQ ID NO:52所示VH的HCDR2第7位丝氨酸残基(对应于SEQ ID NO:52所示的VH第55位)、第9位苏氨酸残基(对应于SEQ ID NO:52所示的VH第57位)、第12位脯氨酸残基(对应于SEQ ID NO:52所示的VH第60位)、第13位天冬氨酸残基(对应于SEQ ID NO:52所示的VH第61位)、第14位丝氨酸残基(对应于SEQ ID NO:52所示的VH第62位)，和对HCDR3的第3位丙氨酸残基(对应于SEQ ID NO:52所示的VH第97位)进行一个或多个氨基酸突变，以及对SEQ ID NO:81所示的VL的LCDR1第11位甘氨酸残基(对应于SEQ ID NO:81所示的VL第29位)进行氨基酸突变。优选地，选择对SEQ ID NO:52所示的VH的HCDR2第12位脯氨酸残基(对应于SEQ ID NO:52所示的VH第60位)、第13位天冬氨酸残基(对应于SEQ ID NO:52所示的VH第61位)、第14位丝氨酸残基(对应于SEQ ID NO:52所示的VH第62位)中的任意一个或两个氨基酸残基，以及选择对HCDR3的第3位丙氨酸残基(对应于SEQ ID NO:52所示的VH第97位)，和对SEQ ID NO:81所示的VL的LCDR1第11位甘氨酸残基(对应于SEQ ID NO:81所示的VL第29位)进行氨基酸突变。
在一些具体实施方案中，本发明的特异性结合HER2胞外区的D1的抗HER2人源化抗体的重链CDR区或轻链CDR区内具有不超过1个、2个、3个、4个、或5个氨基酸突变。
在一个具体实施方案中，将SEQ ID NO:143所示的VH中的CDR区进行1个或多个氨基酸突变，进一步，可以在HCDR1、HCDR2和/或HCDR3进行1个、2个、3个、4个、或5个氨基酸突变。例如，可以在HCDR1的第3位丝氨酸残基(对应于SEQ ID NO:143所示的VH第33位)，在HCDR2的第5位谷氨酸残基(对应于SEQ ID NO:143所示的VH第53位)、第8位谷氨酸残基(对应于SEQ ID NO:143所示的VH第56位)、第14位天冬氨酸残基(对应于SEQ ID NO:143示的VH第62位)、第15位苯丙氨酸残基(对应于SEQ ID NO:143所示的VH第63位)，和/或在HCDR3的第3位精氨酸残基(对应于SEQ ID NO:143所示的VH第97位)、第4位酪氨酸残基(对应于SEQ ID NO:143所示的VH第98位)、第5位天冬氨酸残基(对应于SEQ ID NO:143所示的VH第99位)进行1个或多个氨基酸突变。
在一个具体实施方案中，将SEQ ID NO:171所示的VL中的CDR区进行1个或多个氨基酸突变，例如，可以在LCDR1的第1位赖氨酸残基(对应于SEQ ID NO:171所示的VL第24位)，在LCDR2的第1位丝氨酸残基(对应于SEQ ID NO:171所示的VL第50位)、第4位酪氨酸残基(对应于SEQ ID NO:171所示的VL第53位)、第6位酪氨酸残基(对应于SEQ ID NO:171所示的VL第55位)，和/或在LCDR3的第3位组氨酸残基(对应于SEQ ID NO:171所示的VL第91位)进行1个或多个氨基酸突变。
在一个实施例中，本发明对上述特异性结合HER2胞外区的D1的抗HER2人源化抗体的VH和VL的CDR区进行多个氨基酸突变，以提高本发明抗体的人源化程度、增强抗原结合亲和力、提高表达量，和/或提高稳定性(例如，降低潜在的天冬酰胺脱酰胺的风险)。具体地，对SEQ ID NO:143所示的VH的HCDR2第5位谷氨酸残基(对应于SEQ ID NO:143所示的VH第53位)，以及SEQ ID NO:171所示的VL的LCDR1第1位赖氨酸残基(对应于SEQ ID NO:171所示的VL第24位)，LCDR2第1位丝氨 酸残基(对应于SEQ ID NO:171所示的VL第50位)、第4位酪氨酸残基(对应于SEQ ID NO:171所示的VL第53位)、第6位酪氨酸残基(对应于SEQ ID NO:171所示的VL第55位)和LCDR3第3位组氨酸残基(对应于SEQ ID NO:171所示的VL第91位)进行多个氨基酸突变。
在一些实施方案中，还可以对人源化抗体的可变区的CDR区和框架区进行多个氨基酸突变，以更大程度地改善抗体的一种或多种特性(例如，提高人源化程度、提高抗原结合亲和力、增强稳定性、提高表达量)。在一些具体实施方案中，通过对上述特异性结合HER2胞外区的D1的人源化抗体的SEQ ID NO:143所示的VH的1个或2个CDR区和1个HFR区，和/或SEQ ID NO:171所示VL的3个CDR区进行多个氨基酸突变，优选地，对所述SEQ ID NO:143所示的VH的HCDR2第5位谷氨酸残基(对应于SEQ ID NO:143所示的VH第53位)，HFR3第3位缬氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第68位)、第4位苯丙氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第69位)、第6位亮氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第71位)、第10位缬氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第75位)、第29位苯丙氨酸残基(对应于SEQ ID NO:143所示的VH氨基酸序列第91位)，和/或对所述SEQ ID NO:171所示的LCDR1的第1位赖氨酸残基(对应于SEQ ID NO:171所示的VL第24位)，LCDR2的第1位丝氨酸残基(对应于SEQ ID NO:171所示的VL第50位)、第4位酪氨酸残基(对应于SEQ ID NO:171所示的VL第53位)、第6位酪氨酸残基(对应于SEQ ID NO:171所示的VL第55位)，和在LCDR3第3位组氨酸残基(对应于SEQ ID NO:171所示的VL第91位)进行多个氨基酸突变，不仅使本发明的人源化抗体具备了更高的人源化程度，并且显著提高了与HER2的结合亲和力(KD值约8×10-9～9×10-10M，优选地，5×10-9～9×10-10M)，同时还降低了潜在的天冬酰胺脱酰胺风险，以及提高了表达量。
在另一些实施方案中，本发明提供了分离的抗HER2单克隆抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性地结合HER2胞外区的D3，其包含(1)氨基酸序列为X1YGMS(X1＝S,N和D；即SEQ ID NO:217所示的序列)的HCDR1，优选地，X1＝S；(2)氨基酸序列为SISGX2GX3YX4KYX5X6X7VKG(X2＝G或S；X3＝S或N；X4＝T或A；X5＝P、A、G或V；X6＝D、G、E、P、Q或R；X7＝S、K或N；即SEQ ID NO:218所示的序列)的HCDR2，优选地，X2＝G，X3＝S，X4＝T，X5＝P或V，X6＝D、P或E，X7＝S或K；(3)氨基酸序列为DYX8GFFDV(X8＝A、I、N、R、S、或V；即SEQ ID NO:219所示的序列)的HCDR3，优选地，X8＝V；(4)氨基酸序列为RSSQSLX9X10SNX11NTYLH(X9＝V或L；X10＝H或S；X11＝G、A、I、S、R或T；即SEQ ID NO:220所示的序列)的LCDR1，优选地，X9＝V或L，X10＝H，X11＝R；(5)氨基酸序列为KVSNRX12S(X12＝F、D或P；即SEQ ID NO:221所示的序列)的LCDR2，优选地，X12＝F；(6)氨基酸序列为X13QSTHVPX14T(X13＝S或Q；X14＝Y或W；即SEQ ID NO:222所示的序列)的LCDR3，优选地，X13＝S，X14＝Y，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。所述抗体或其抗原结合片段人源化程度高、表达量高，并且消除了天冬氨酸异构化的风险。
在一些具体的实施方案中，上述特异性结合HER2胞外区的D3的抗体或其抗原结合片段包含：如SEQ ID NO:11、12、或13所示的HCDR1；如SEQ ID NO:14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、或31所示的HCDR2；和如SEQ ID NO:32、33、34、35、36、或37所示的HCDR3；以及如SEQ ID NO:38、39、40、41、42、43、44、或45所示的LCDR1；如SEQ ID NO:46、47、或48所示的LCDR2；和如SEQ ID NO:49、50、或51所示的LCDR3。
在一个具体的实施方案中，本发明的抗HER2抗体或其抗原结合片段特异性地结合HER2胞外区的D3，其包含如下的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：
(1)如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:14、15、16、17、18、19、或20所示的HCDR2，如SEQ ID NO:32所示的HCDR3；以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3；或
(2)如SEQ ID NO:12或13所示的HCDR1、如SEQ ID NO:14所示的HCDR2、如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3；或
(3)如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:14所示的HCDR2、如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:47或48所示的LCDR2、如SEQ ID NO:49所示的LCDR3；或
(4)如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:14所示的HCDR2、如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:39、40、41、42、43、44、或45所示的LCDR1、如SEQ ID NO:46所示的 LCDR2、如SEQ ID NO:49所示的LCDR3；或
(5)如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:14所示的HCDR2、如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:50或51所示的LCDR3；或
(6)如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:14所示的HCDR2，如SEQ ID NO:33、34、35、36、或37所示的HCDR3；以及如SEQ ID NO:38所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3；或
(7)如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:14、21、22、23、24、25、26、27、28、29、30、或31所示的HCDR2，如SEQ ID NO:37所示的HCDR3；以及如SEQ ID NO:42所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3；或
(8)如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:21所示的HCDR2、如SEQ ID NO:32所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3。
在一个具体的实施方案中，本发明的抗HER2抗体或其抗原结合片段特异性地结合HER2胞外区的D3，包含：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:14、17、19、21、23、24、27、28、29、30、或31所示的HCDR2，如SEQ ID NO:32或37所示的HCDR3，以及如SEQ ID NO:38或42所示的LCDR1，如SEQ ID NO:46所示的LCDR2，如SEQ ID NO:49所示的LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。优选地，所述抗体或其抗原结合片段包含：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:14、17、19、24、29、30、或31所示的HCDR2，如SEQ ID NO:32或37所示的HCDR3，以及如SEQ ID NO:38或42所示的LCDR1、如SEQ ID NO:46所示的LCDR2、如SEQ ID NO:49所示的LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。更优选地，所述抗体或其抗原结合片段包含：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:24、29、30、或31所示的HCDR2和如SEQ ID NO:37所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1，如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在另一些实施方案中，本发明提供了分离的抗HER2单克隆抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性地结合HER2胞外区的D1，其包含(1)氨基酸序列为DYX15MH(X15＝S或A；即SEQ ID NO:223所示的序列)的HCDR1，优选地，X15＝S；(2)氨基酸序列为WINTX16TGX17PTYADX18X19KG(X16＝E、N、G、Y或I；X17＝E、D或S；X18＝D、K或N；X19＝F或V；即SEQ ID NO:224所示的序列)的HCDR2，优选地，X16＝G，X17＝E，X18＝D，X19＝F；(3)氨基酸序列为VGX20X21X22YAMDY(X20＝R或Y；X21＝Y或G；X22＝D或S；即SEQ ID NO:225所示的序列)的HCDR3，优选地，X20＝R，X21＝Y，X22＝D；(4)氨基酸序列为X23ASQDVYTAVA(X23＝K或R；即SEQ ID NO:226所示的序列)的LCDR1，优选地，X23＝R；(5)氨基酸序列为X24ASX25RX26T(X24＝S、A、D、E、K、L、Q、R、W或Y；X25＝Y、D、E、K、N、Q、S或T；X26＝Y、A、E、P或Q；即SEQ ID NO:227所示的序列)的LCDR2，优选地，X24＝S、A、L或Y，X25＝S，X26＝P；(6)氨基酸序列为QQX27YSTPPT(X27＝H、S、A或Y；即SEQ ID NO:228所示的序列)的LCDR3，优选地，X27＝Y，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。所述抗体或其抗原结合片段不仅对HER2具有高亲和力(例如，KD值＜1×10-8M，或＜5×10-9M，或＜1×10-9M，优选地，KD值＜5×10-9M，或＜1×10-9M)，而且具有更高的人源化程度以及稳定性。
在一些具体的实施方案中，上述特异性结合HER2胞外区的D1的抗体或其抗原结合片段包含：如SEQ ID NO:98或99所示的HCDR1；如SEQ ID NO:100、101、102、103、104、105、106、107、108、或109所示的HCDR2；如SEQ ID NO:110、111、112、或113所示的HCDR3；以及如SEQ ID NO:114或115所示的LCDR1；如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、或138所示的LCDR2；如SEQ ID NO:139、140、141、或142所示的LCDR3。
在一个具体的实施方案中，本发明的抗HER2抗体或其抗原结合片段特异性地结合HER2胞外区的D1，其包含如下的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：
(1)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:100所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：114所示的LCDR1，如SEQ ID NO:116、117、118、119、120、121、122、123、124、125、126、127、或128所示的LCDR2，如SEQ ID NO:139所示的LCDR3；或
(2)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:100所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：114所示的LCDR1，如SEQ ID NO:116所示的LCDR2，如SEQ ID NO:140、141、或142所示的LCDR3；或
(3)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:101、102、103、104、105、106、107、108、或109所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：114所示的LCDR1，如SEQ ID NO:116所示的LCDR2，如SEQ ID NO:139所示的LCDR3；或
(4)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:100所示的HCDR2，如SEQ ID NO:111、112、或113所示的HCDR3，如SEQ ID NO：114所示的LCDR1，如SEQ ID NO:116所示的LCDR2，如SEQ ID NO:139所示的LCDR3；或
(5)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:100所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：115所示的LCDR1，如SEQ ID NO:116所示的LCDR2，如SEQ ID NO:139所示的LCDR3；或
(6)如SEQ ID NO:99所示的HCDR1，如SEQ ID NO:100所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：114所示的LCDR1，如SEQ ID NO:116所示的LCDR2，如SEQ ID NO:139所示的LCDR3；或
(7)如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：115所示的LCDR1，如SEQ ID NO:129、130、131、132、133、134、135、136、137、或138所示的LCDR2，如SEQ ID NO:142所示的LCDR3。
在一个具体的实施方案中，本发明的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区的D1，其包含如下HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:100或102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：114或115所示的LCDR1，如SEQ ID NO:116、123、127、129、130、131、132、133、134、135、136、137、或138所示的LCDR2，如SEQ ID NO:139或142所示的LCDR3。优选地，所述抗体或其抗原结合片段包含：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，如SEQ ID NO：115所示的LCDR1，如SEQ ID NO:129、133、136、或138所示的LCDR2，如SEQ ID NO:142所示的LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。所述抗体或其抗原结合片段与HER2的结合亲和力(KD)值＜5×10-9M，或＜1×10-9M，优选＜1×10-9M。更优选地，所述抗体或其抗原结合片段包含：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2和如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:115所示的LCDR1，如SEQ ID NO:133所示的LCDR2和如SEQ ID NO:142所示的LCDR3，或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施方案中，本发明的抗HER2抗体或其抗原结合片段进一步包含具有所述HCDR1、HCDR2和HCDR3的重链可变区以及具有LCDR1、LCDR2和LCDR3的轻链可变区。
在一些具体实施方案中，本发明的抗体或其抗原结合片段可特异性地结合HER2胞外区的D3，其包含：与SEQ ID NO:52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、或80所示的氨基酸序列具有至少80％(例如至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％)同一性的重链可变区VH，以及与SEQ ID NO:81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、或97所示的氨基酸序列具有至少80％(例如至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％)同一性的轻链可变区VL。
在一个具体实施方案中，本发明的抗体或其抗原结合片段可特异性地结合HER2胞外区的D3，其包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和具有所述LCDR1、LCDR2和LCDR3的轻链可变区，所述重链可变区和轻链可变区各自包括分别与SEQ ID NO:52所示的VH和SEQ ID NO:81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、或97所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者分别与SEQ ID NO:53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、或68所 示的VH和SEQ ID NO:81所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者分别与SEQ ID NO:68、69、70、71、72、73、74、75、76、77、78、79、或80所示的VH和SEQ ID NO:92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施例中，所述的特异性地结合HER2胞外区的D3的抗体或其抗原结合片段包含：分别与SEQ ID NOs:52、57、62、68、73、78、79和80中的任一者所示的VH，以及SEQ ID NO:81或92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。优选地，所述抗体或其抗原结合片段包含：分别与SEQ ID NO:73、78、79、或80所示的VH和SEQ ID NO:92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列，更优选的是分别与SEQ ID NO:80所示的VH和SEQ ID NO:92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，本发明的抗体或其抗原结合片段可特异性地结合HER2胞外区的D1，其包含：与SEQ ID NO:143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、或170所示的氨基酸序列具有至少80％(例如至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％)同一性的重链可变区VH，以及与SEQ ID NO:171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、或198所示的氨基酸序列具有至少80％(例如至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％)同一性的轻链可变区VL。
在一个具体实施方案中，本发明的抗体或其抗原结合片段可特异性地结合HER2胞外区的D1，其包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和具有所述LCDR1、LCDR2和LCDR3的轻链可变区，其包括分别与SEQ ID NO:143所示的VH和SEQ ID NO:171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、或188所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者分别与SEQ ID NO:144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、或170所示的VH和SEQ ID NO:171所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施例中，所述特异性地结合HER2胞外区的D1的抗体或其抗原结合片段包含：分别与SEQ ID NOs:143、148、156、158、160、161、165和169中的任一者所示的VH，以及SEQ ID NO:171、172、180、184、188、189、190、191、192、193、194、195、196、197、或198中的任一者所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。优选地，所述的抗体或其抗原结合片段包含：分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列，更优选的是分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、193、196、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。进一步优选地，本发明的上述的抗体或其抗原结合片段包含：SEQ ID NO:169所示的VH，以及SEQ ID NO:193所示的VL。
本发明的抗HER2人源化抗体或其抗原结合片段与表达HER2的细胞有较强的结合活性，且对HER2介导的信号转导通路不具有影响作用，不与人源ErbB/HER家族其它成员(包括EGFR、HER3和HER4)发生交叉结合反应，具有较高的人源化程度和稳定性。
2、抗HER2互补性双特异抗体
一方面，本发明提供了一种抗HER2互补性双特异抗体，所述互补性双特异抗体包含两种能够非竞争性地同时结合HER2胞外区的抗原结合结构域，其中第一抗原结合结构域和第二抗原结合结构域各自特异性地结合HER2胞外区的不同表位。进一步，所述互补性双特异抗体可以同时结合至HER2胞外区的D1、D3和D4中的任意两个表位，优选地，可同时结合至HER2胞外区的D1和D3，例如，其中第一抗原结合结构域与HER2胞外区的D3特异性结合，第二抗原结合结构域与HER2胞外区的D1特异性结合，所述第一和第二抗原结合结构域的结合表位均不同于Trastuzumab或Pertuzumab的抗原结合表位。
本发明的抗HER2互补性双特异抗体具有以下功能特性：
(i)所述抗HER2互补性双特异抗体是一个四价分子，其可以同时结合HER2胞外区的两个不同表位，因此能够交联细胞表面的HER2而形成抗原-抗体交联聚集体或者簇，显而易见地，该聚集体或簇形成的效率及大小受制于细胞表面靶抗原的丰度，并且簇的形成会引发细胞对其的内吞反应，该内吞反应的速率与强度与簇的大小成正相关。具体地，相较于对应的单特异性抗HER2抗体或其抗原结合片段以及Trastuzumab，所述互补性双特异抗体具有显著增强的内化效率，其在HER2过表达肿瘤细胞中的内吞率高达80％以上，在HER2低表达肿瘤细胞中的内吞率高达60％以上。
(ii)不同于对应的单特异性抗HER2抗体或其抗原结合片段以及Trastuzumab在内化后的胞内转运途径以重循环至细胞表面(Recycling)为主，所述互补性双特异抗体与HER2在细胞表面形成簇并内化后，溶酶体转运(Lysosomal Trafficking)为其占绝对主导地位的胞内转运途径，因此所述互补性双特异抗体与HER2形成的簇能够被高效地转运入溶酶体而降解。
(iii)所述互补性双特异抗体由于能够有效地引发肿瘤细胞表面HER2的降解，因而能够显著抑制HER2过表达肿瘤细胞的增殖。
(iv)所述互补性双特异抗体不影响HER2及其介导的下游信号转导通路的调控，包括不诱导、不阻断、不抑制配体(例如，NRG-1)诱导的HER2二聚化及其下游信号转导通路的激活，因此所述抗HER2互补性双特异抗体不影响正常组织或细胞(例如，心肌细胞)中HER2及其介导的下游信号转导通路的正常生物学功能与调控。
(v)所述抗HER2互补性双特异抗体具有较高的稳定性和表达量，以及较高的单体含量(约98％以上)。
所述互补性双特异抗体的形式包括但不限于，基于scFv或Diabody的双特异性形式(例如，scFv-scFv或scFv-Fab或Fab-scFv)、IgG-scFv融合蛋白、DVD-Ig、四源杂交瘤(Quadroma)、凸起-进入-孔洞(Knob-into-hole)、共同轻链(Common Light Chain)、CrossMab、CrossFab、SEEDbody、亮氨酸拉链(Leucine Zipper)、Duobody、IgG1/IgG2、双重作用(Dual-acting)Fab(DAF)-IgG和Mab2双特异性形式(参见，例如，Klein等,mAbs 2012,4:653-663及其引用的参考文献)。本发明示例性抗HER2互补性双特异抗体采用DVD-Ig形式，其不仅对表达HER2肿瘤细胞的结合活性较高，而且表现出较高的细胞内化率，此外，还能保证本发明的互补性双特异抗体的稳定性(例如，单体含量较高，约98％以上)。
所述抗HER2互补性双特异抗体与表达HER2的细胞具有较强的结合活性，且与人源ErbB/HER家族其它成员(包括EGFR、HER3和HER4)无交叉结合活性。
在表达HER2的肿瘤细胞中，所述抗HER2互补性双特异抗体表现出显著增强的内化效率。在一些实施方案中，所述互补性双特异抗体相较于对应的单特异性抗HER2抗体或其抗原结合片段以及Trastuzumab，在HER2过表达(IHC 3+、或IHC 2+/FISH+)的肿瘤细胞中的内吞率高达80％以上。在一些实施方案中，所述互补性双特异抗体相较于对应的单特异性抗HER2抗体或其抗原结合片段以及Trastuzumab，在HER2低表达(IHC 2+/FISH-、或IHC 1+)的肿瘤细胞中的内吞率高达60％。
所述抗HER2互补性双特异抗体能够有效促使HER2在肿瘤细胞中的降解。在一些实施方案中，通过Western-blot方法检测发现所述互补性双特异抗体能够诱导HER2过表达肿瘤细胞(例如，BT474细胞)的HER2的降解，而与所述互补性双特异抗体相对应的单特异性抗HER2抗体或其抗原结合片段以及Trastuzumab则不能诱发HER2在所述肿瘤细胞内的降解。进一步，所述抗HER2互补性双特异抗体能够有效地降低肿瘤细胞表面HER2的表达丰度，因此所述抗HER2互补性双特异抗体能够显著抑制HER2过表达肿瘤细胞的增殖。在一些实施方案中，所述抗HER2互补性双特异抗体对HER2过表达的肿瘤细胞(例如，BT474细胞)的增殖具有显著抑制作用，其抑制活性与Trastuzumab的相当，而与所述互补性双特异抗体相对应的单特异性抗HER2抗体或其抗原结合片段则不能抑制肿瘤细胞的增殖。
所述抗HER2互补性双特异抗体对HER2介导的信号转导通路没有影响作用，保持了相对应的单特异性抗HER2抗体或其抗原结合片段不影响HER2生物学功能的特性，因此对正常组织或细胞(例如，心肌细胞)中HER2的正常生物学功能不会产生不良影响。在一些实施方案中，所述抗HER2互补性双特异抗体不诱导、不阻断、不抑制配体(例如，NRG-1)诱导的HER2二聚化及其介导的下游信号通路中的AKT的磷酸化和/或去磷酸化。由此，不同于现有靶向HER2的治疗剂(例如基于Trastuzumab和/或Pertuzumab的治疗剂)，所述抗HER2互补性双特异抗体潜在的诱发心脏毒副作用的风险极低。
另一方面，本发明的抗HER2互补性双特异抗体，其第一和第二抗原结合结构域可以衍生自本发明的任何两个互相不竞争性结合HER2的单特异性抗HER2抗体或其抗原结合片段，也可衍生自目前已知的抗HER2抗体或其抗原结合片段。
本发明的示例性抗HER2互补性双特异抗体可基于表2所示的特异性结合HER2胞外区的D3和表3所示的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段构建得到。本发明的示例性抗HER2互补性双特异抗体包含两种不同的抗原结合结构域，其中第一抗原结合结构域包含表2所示的特异性结合HER2胞外区的D3的抗HER2抗体或其抗原结合片段的任意的至少一个CDR区和/或任一个可变 区，第二抗原结合结构域包含表3所示的特异性结合HER2胞外区的D1的抗HER2抗体或其抗原结合片段的任意的至少一个CDR区和/或任一个可变区。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一或第二抗原结合结构域包含重链可变区CDRs和/或轻链可变区CDRs，所述第一抗原结合结构域的重链可变区CDRs包含表2中所列出的重链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或分别与HCDR1、HCDR2和HCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第一抗原结合结构域的轻链可变区CDRs包含表2中所列出的轻链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或分别与LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的重链可变区CDRs包含表3中所列出的重链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或分别与HCDR1、HCDR2和HCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的轻链可变区CDRs包含表3中所列出的轻链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或分别与LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，所述抗HER2互补性双特异抗体的第一抗原结合结构域特异性地结合HER2胞外区的D3，包含：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:21、23、24、27、28、29、30、或31所示的HCDR2，如SEQ ID NO:37所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1，如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3，或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一个具体实施方案中，所述抗HER2互补性双特异抗体的第一抗原结合结构域特异性地结合HER2胞外区的D3，包含：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:24、29、30、或31所示的HCDR2，如SEQ ID NO:37所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1，如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3，或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，所述抗HER2互补性双特异抗体的第二抗原结合结构域特异性地结合HER2胞外区的D1，包含：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:115所示的LCDR1，如SEQ ID NO:129、130、131、132、133、134、135、136、137、或138所示的LCDR2和如SEQ ID NO:142所示的LCDR3，或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一个具体实施方案中，所述抗HER2互补性双特异抗体的第二抗原结合结构域特异性地结合HER2胞外区的D1，包含：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:115所示的LCDR1，如SEQ ID NO:133所示的LCDR2和如SEQ ID NO:142所示的LCDR3，或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一或第二抗原结合结构域进一步包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和/或具有LCDR1、LCDR2和LCDR3的轻链可变区，所述第一抗原结合结构域的重链可变区包含表2中所列出的VH氨基酸序列中任一个的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第一抗原结合结构域的轻链可变区包含表2中所列出的VL氨基酸序列中任一个的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的重链可变区包含表3中所列出的VH氨基酸序列中任一个的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的轻链可变区包含表3中所列出的VL氨基酸序列中任一个的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，所述抗HER2互补性双特异抗体的第一抗原结合结构域特异性地结合HER2胞外区的D3，包含：如SEQ ID NO:70、72、73、76、77、78、79、或80所示的VH，以及如SEQ ID NO: 92所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一个具体实施方案中，所述抗HER2互补性双特异抗体的第一抗原结合结构域特异性地结合HER2胞外区的D3，包含：如SEQ ID NO:73、78、79、或80所示的VH，以及如SEQ ID NO:92所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，所述抗HER2互补性双特异抗体的第二抗原结合结构域特异性地结合HER2胞外区的D1，包含：如SEQ ID NO:169所示的VH，以及如SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一个具体实施方案中，所述抗HER2互补性双特异抗体的第二抗原结合结构域特异性地结合HER2胞外区的D1，包含：如SEQ ID NO:169所示的VH，以及如SEQ ID NO:193所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些具体实施方案中，所述抗HER2互补性双特异抗体包含第一和第二抗原结合结构域，所述第一抗原结合结构域包含如下重链可变区CDRs和轻链可变区CDRs，或分别与所述各CDR具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:21、23、24、27、28、29、30、或31所示的HCDR2，如SEQ ID NO:37所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1，如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3；以及所述第二抗原结合结构域包含如下重链可变区CDRs和轻链可变区CDRs，或分别与所述各CDR具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:115所示的LCDR1，如SEQ ID NO:129、130、131、132、133、134、135、136、137、或138所示的LCDR2和如SEQ ID NO:142所示的LCDR3。
在一个具体实施方案中，所述抗HER2互补性双特异抗体包含第一和第二抗原结合结构域，所述第一抗原结合结构域包含如下重链可变区CDRs和轻链可变区CDRs，或分别与所述的各CDR具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：如SEQ ID NO:11所示的HCDR1，如SEQ ID NO:24、29、30、或31所示的HCDR2，如SEQ ID NO:37所示的HCDR3，以及如SEQ ID NO:42所示的LCDR1，如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:49所示的LCDR3，以及所述第二抗原结合结构域包含如下重链可变区CDRs和轻链可变区CDRs，或分别与所述各CDR具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列：如SEQ ID NO:98所示的HCDR1，如SEQ ID NO:102所示的HCDR2，如SEQ ID NO:110所示的HCDR3，以及如SEQ ID NO:115所示的LCDR1，如SEQ ID NO:133所示的LCDR2和如SEQ ID NO:142所示的LCDR3。
在一些具体实施方案中，所述抗HER2互补性双特异抗体包含第一和第二抗原结合结构域，所述第一抗原结合结构域包含重链可变区VH以及轻链可变区VL，所述重链可变区VH和轻链可变区VL包含如SEQ ID NO:70、72、73、76、77、78、79、或80所示的VH氨基酸序列，以及SEQ ID NO:92所示的VL氨基酸序列，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列，以及所述第二抗原结合结构域包含重链可变区VH以及轻链可变区VL，所述重链可变区VH和轻链可变区VL包含如SEQ ID NO:169所示的VH氨基酸序列和SEQ ID NO:193所示的VL氨基酸序列，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一个具体实施方案中，所述抗HER2互补性双特异抗体包含第一和第二抗原结合结构域，所述第一抗原结合结构域包含重链可变区VH以及轻链可变区VL，所述重链可变区VH和轻链可变区VL包含如SEQ ID NO:73、78、79、或80所示的VH氨基酸序列，以及SEQ ID NO:92所示的VL氨基酸序列，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列，以及所述第二抗原结合结构域包含重链可变区VH以及轻链可变区VL，所述重链可变区VH和轻链可变区VL包含如SEQ ID NO:169所示的VH氨基酸序列和SEQ ID NO:193所示的VL氨基酸序列，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
本发明的示例性抗HER2互补性双特异抗体还可以基于本发明所述的特异性结合HER2胞外区的D1 和D4的抗HER2抗体或其抗原结合片段构建得到。本发明的示例性抗HER2互补性双特异性抗体包含第一和第二抗原结合结构域，且所述第一和第二抗原结合结构域能够彼此之间非竞争地同时结合HER2，其中第一抗原结合结构域包含SEQ ID NO:7所示的VH氨基酸序列和SEQ ID NO:8所示的VL氨基酸序列，或者，所述第一抗原结合结构域包含SEQ ID NO:9所示的VH氨基酸序列和SEQ ID NO:10所示的VL氨基酸序列，或者，所述第一抗原结合结构域包含表3所示的抗HER2抗体或其抗原结合片段的任意的至少一个CDR区和/或任一个可变区，并能特异性地结合HER2胞外区的D1；第二抗原结合结构域包含SEQ ID NO:5所示的VH氨基酸序列和SEQ ID NO:6所示的VL氨基酸序列，并能特异性地结合HER2胞外区的D4。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一或第二抗原结合结构域包含重链可变区CDRs和/或轻链可变区CDRs，所述第一抗原结合结构域的重链可变区CDRs包含SEQ ID NO:7所示的VH氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:205、206和207所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；所述第一抗原结合结构域的轻链可变区CDRs包含SEQ ID NO:8所示的VL氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:208、209和210所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；或者，所述第一抗原结合结构域的重链可变区CDRs包含SEQ ID NO:9所示的VH氨基酸序列中的一个、或两个、或三个CDR或如SEQ ID NOs:211、212和213所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；所述第一抗原结合结构域的轻链可变区CDRs包含SEQ ID NO:10所示的VL氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:214、215和216所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；或者，所述第一抗原结合结构域的重链可变区CDRs包含表3中所列出的重链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第一抗原结合结构域的轻链可变区CDRs包含表3中所列出的轻链可变区CDRs中任一个、两个或三个CDR区的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的重链可变区CDRs包含SEQ ID NO:5所示的VH氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:199、200和201所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；所述第一抗原结合结构域的轻链可变区CDRs包含SEQ ID NO:6所示的VL氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:202、203和204所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体。
本发明的示例性抗HER2互补性双特异抗体还可以基于本发明所述的特异性结合HER2胞外区的D3和D4的抗HER2抗体或其抗原结合片段构建得到。本发明的示例性抗HER2互补性双特异性抗体包含第一和第二抗原结合结构域，且所述第一和第二抗原结合结构域能够彼此之间非竞争地同时结合HER2，其中第一抗原结合结构域包含表2所示的特异性结合HER2胞外区的D3的抗HER2抗体或其抗原结合片段的任意的至少一个CDR区和/或任一个可变区；第二抗原结合结构域包含SEQ ID NO:5所示的VH氨基酸序列和SEQ ID NO:6所示的VL氨基酸序列，并能特异性地结合HER2胞外区的D4。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一或第二抗原结合结构域包含重链可变区CDRs和/或轻链可变区CDRs，所述第一抗原结合结构域的重链可变区CDRs包含表2中所列出的重链可变区CDRs中任一个、两个、或三个CDR区的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第一抗原结合结构域的轻链可变区CDRs包含表2中所列出的轻链可变区CDRs中任一个、两个、或三个CDR区的氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域的重链可变区CDRs包含SEQ ID NO:5所示的VH氨基酸序列中的一个、两个、或三个的CDR或如SEQ ID NOs:199、200和201所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体；所述第一抗原结合结构域的轻链可变区CDRs包含SEQ ID NO:6所示的VL氨基酸序列中的一个、两个、或三个CDR或如SEQ ID NOs:202、203和204所示的氨基酸序列或其变异体，所述变异体包括人源化抗体或任何其它变异体。
另一方面，本发明的示例性抗HER2互补性双特异抗体可采用DVD-Ig形式进行构建(参见US7612181)，所述抗HER2互补性双特异抗体的第一或第二抗原结合结构域可以是Fv形式/结构域，也可以是Fab或IgG形式/结构域，即，如果第一抗原结合结构域是Fv，那么第二抗原结合结构域则是Fab或IgG；如果第二抗原结合结构域是Fv，那么第一抗原结合结构域则是Fab或IgG。所述Fv结构域通过连接子序列融合到或可操作地连接到所述Fab或IgG结构域，所述连接子不仅具有低免疫原性，而且还能保证所述互补性双特异抗体的稳定性，优选柔性肽作为连接子。
如本文所使用的，术语“连接子”是指连接两个化合物，如两个多肽分子(包括但不限于未修饰或经修饰的氨基酸或氨基酸序列)。连接子可以由1个或多个连接分子构成，或者可以包括连接分子和至少一 个旨在将连接分子和化合物分开特定距离的间隔区分子。
术语“可操作地连接”是指具有不同功能特性的氨基酸序列、肽或蛋白质的连接，如通过本文所述的连接子序列将Fv结构域与Fab或IgG结构域的连接。
在一些实施方案中，所述抗HER2互补性双特异抗体的第一抗原结合结构域特异性地结合HER2胞外区的D3，且为Fv结构域，所述第一抗原结合结构域包含表2所示的抗HER2抗体或其抗原结合片段的重链可变区CDRs和轻链可变区CDRs，和/或重链可变区VH和轻链可变区VL；所述抗HER2互补性双特异抗体的第二抗原结合结构域特异性地结合HER2胞外区的D1，且为Fab结构域或IgG结构域，所述第二抗原结合结构域包含表3所示的抗HER2抗体或其抗原结合片段的重链可变区CDRs和轻链可变区CDRs，和/或重链可变区VH和轻链可变区VL；所述Fv结构域中的VH结构域C-末端通过连接子序列融合到或可操作地连接到所述Fab或IgG结构域中的VH结构域N-末端，所述Fv结构域中的VL结构域C-末端通过连接子序列融合到或可操作地连接到所述Fab或IgG结构域中的VL结构域N-末端。
在一些具体实施方案中，所述连接子序列包括不同拷贝数的GGGGS(G4S)序列，例如1个、2个、3个、4个、或5个拷贝。所述抗HER2互补性双特异抗体Fv结构域中的VH结构域与所述互补性双特异抗体Fab或IgG结构域中的VH结构域连接的连接子G4S的拷贝数可以和所述互补性双特异抗体Fv结构域中的VL结构域与所述互补性双特异抗体Fab或IgG结构域中的VL结构域连接的连接子G4S拷贝数相同或者不同，优选地，Fv结构域与Fab或IgG结构域之间VH结构域连接的连接子G4S拷贝数与二者之间VL结构域连接的连接子G4S拷贝数不同，其中二者VH结构域之间连接的连接子拷贝数优选1个，二者VL结构域之间连接的连接子拷贝数优选3个，由此可以有效保证本发明的抗HER2互补性双特异抗体的结构稳定性以及提高在表达HER2肿瘤细胞中的内吞率。
在一个具体实施方案中，所述抗HER2互补性双特异抗体包含第一和第二抗原结合结构域，其中，第一抗原结合结构域包含表2所示的抗HER2抗体或其抗原结合片段的VH和VL，第二抗原结合结构域包含表3所示的抗HER2抗体或其抗原结合片段的VH和VL，所述第一抗原结合结构域为Fv结构域，第二抗原结合结构域为Fab或IgG结构域，所述Fv结构域中的VH结构域C-末端通过连接子(例如(G4S)n，n为大于0、如1-3的整数；优选(G4S)1连接到所述Fab或IgG结构域中的VH结构域N-末端，所述Fv结构域中的VL结构域C-末端通过连接子(例如(G4S)n，n为1-3的整数；优选(G4S)3)连接到所述Fab或IgG结构域中的VL结构域N-末端。
另一方面，本发明的抗HER2互补性双特异抗体还可以包含恒定区，所述恒定区包括抗体重链恒定区和轻链恒定区。本发明的重链恒定区包括人IgG重链恒定区Fc区的天然和突变蛋白形式，还包括含有促进二聚体形成的铰链区的多肽截短形式。在一些实施方案中，Fc区包含抗体CH2和CH3域。包含Fc部分的融合蛋白(和由此形成的低聚物)提供了容易通过蛋白A(Protein A)或蛋白G(Protein G)进行亲和色谱法纯化的优点，以及延长血清半衰期。优选的Fc区来源于人IgG，包括IgG1、IgG2、IgG3和IgG4。在本文中，Fc区的特定氨基酸残基的位置是依据EU编号系统来确定的。
抗体的Fc区的一个功能是在抗体结合其靶分子时与免疫系统产生“效应子功能”，包括产生抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)和/或补体依赖性细胞毒性(CDC)。ADCC和ADCP通过Fc与免疫细胞表面上Fc受体(FcR)的结合来介导(Raghavan等,Annu Rev Cell Dev Biol 1996,12:181-220；Ghetie等,Annu Rev Immunol 2000,18:739-766；Ravetch等,Annu Rev Immunol 2001,19:275-290)，所述的免疫细胞包括单核细胞、巨噬细胞、嗜中性粒细胞、树突细胞、嗜曙红细胞、肥大细胞、血小板、B细胞、大颗粒淋巴细胞、朗格汉斯细胞、NK细胞和T细胞。CDC通过Fc与例如C1q等补体系统的蛋白质结合来介导(Ward等,Ther Immunol 1995,2:77-94)。
在一些实施方案中，本发明的抗HER2互补性双特异抗体包含经工程化改造的IgG Fc区，以降低Fc介导的效应子功能。具有降低了的效应子功能的示例性抗体包括含有以下氨基酸突变的Fc区：
N297A或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)
优选的工程化的Fc区是具有L234F/L235E/P331S(EU编号系统)氨基酸取代的人IgG1Fc，能够降低Fc区与一个或多个FcγR和C1q的结合(Oganesyan等,Acta Crystallogr D Biol Crystallogr 2008,64:700-704；US5624821；US6194551)。FcγR蛋白家族包括FcγRI(又称为CD64)，包括同工型FcγRIa、FcγRIb和FcγRIc；FcγRII(又称为CD32)，包括同工型FcγRIIa、FcγRIIb和FcγRIIc；以及FcγRIII(又称为CD16)， 包括同工型FcγRIIIa和FcγRIIIb(Jefferis等,Immunol Lett 2002,82:57-65)。其中，FcγRI、FcγRIIa、FcγRIIc以及FcγRIIIa可诱导ADCC、内吞作用、吞噬作用和/或细胞因子释放。结合性质包括但不限于结合特异性、结合亲和力(KD)、以及解离和结合速率(分别为kdis和ka)，本领域技术人员可通过任意一种或多种结合性质来分析工程化的Fc区是否具有改变了的ADCC和/或CDC活性。在一些实施方案中，本发明的抗HER2互补性双特异抗体中的重链恒定区和轻链恒定区可为来自人IgG1或IgG4的重链恒定区(可为天然或突变的，如以SEQ ID NO:231示出的野生型人IgG1恒定区)，和人Kappa(κ)链或Lambda(λ)恒定区。
在一些具体实施方案中，所述抗HER2互补性双特异抗体的重链恒定区包含L234F/L235E/P331S(EU编号系统)氨基酸取代的人IgG1Fc(如SEQ ID NO:232所示)，其具有对一个或多个FcγR(例如，FcγRI、FcγRIIa、FcγRIIb、FcγRIIIa和FcγRIIIa)以及C1q降低了的结合亲和力。在一些实施例中，在所述抗HER2互补性双特异抗体的重链恒定区中引入L234F/L235E/P331S突变使得所述互补性双特异抗体与FcγRI、FcγRIIa(167H)、FcγRIIb、FcγRIIIa(176V)和/或FcγRIIIa(176F)基本不结合。在一些实施例中，在所述抗HER2互补性双特异抗体的重链恒定区中引入L234F/L235E/P331S突变使得所述互补性双特异抗体与C1q基本不结合。在一些实施例中，在所述抗HER2互补性双特异抗体的重链恒定区中引入L234F/L235E/P331S突变不会影响所述互补性双特异抗体与FcRn的结合亲和力。在一些实施例中，在所述抗HER2互补性双特异抗体的重链恒定区中引入L234F/L235E/P331S突变可显著降低所述互补性双特异抗体的ADCC活性。在一个实施例中，相较于Fc区不含有所述氨基酸突变的抗体，所述互补性双特异抗体的ADCC活性显著减弱或检测不到，ADCC活性的减弱或消除可能是由所述的互补性双特异抗体对FcγR的结合亲和力显著降低所致。
在一些具体实施方案中，所述抗HER2互补性双特异抗体包含轻链恒定区，所述轻链恒定区选自Kappa恒定区(如SEQ ID NO:233所示)。
在一方面，本发明的抗HER2互补性双特异抗体包含4条多肽链，其中两条多肽链包含VH1-L1-VH2-C-(Fc)n，VH1表示特异性结合HER2胞外区的D3的第一抗原结合结构域的重链可变区，L1表示连接子，VH2表示特异性结合HER2胞外区的D1的第二抗原结合结构域的重链可变区，C表示重链恒定区CH1，Fc表示重链恒定区Fc结构域，n是0或1；以及另外两条多肽链包含VL1-L2-VL2-CL，其中VL1表示特异性结合HER2胞外区的D3的第一抗原结合结构域的轻链可变区，L2表示连接子，VL2表示特异性结合HER2胞外区的D1的第二抗原结合结构域的轻链可变区，CL是IgG轻链恒定区。
在一些实施方案中，所述VH1包含如SEQ ID NO:11所示的HCDR1氨基酸序列，如SEQ ID NO:24、29、30、或31所示的HCDR2氨基酸序列和如SEQ ID NO:37所示的HCDR3氨基酸序列，或分别与所述的HCDR1、HCDR2和HCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；L1包含如SEQ ID NO:229所示的连接子氨基酸序列；VH2包含如SEQ ID NO:98所示的HCDR1氨基酸序列，如SEQ ID NO:102所示的HCDR2氨基酸序列和如SEQ ID NO:110所示的HCDR3氨基酸序列，或分别与所述的HCDR1、HCDR2和HCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；Fc包含工程化的Fc结构域，包括具有L234F/L235E/P331S(EU编号系统)氨基酸取代的人IgG1Fc，其氨基酸序列如SEQ ID NO:232所示。
在一些具体实施方案中，所述VH1包含如SEQ ID NO:73、78、79、或80所示的VH氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述VH2包含如SEQ ID NO:169所示的VH氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施方案中，所述VL1包含如SEQ ID NO:42所示的LCDR1氨基酸序列，如SEQ ID NO:46所示的LCDR2氨基酸序列和如SEQ ID NO:49所示的LCDR3氨基酸序列，或分别与所述的LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；L2包含如SEQ ID NO:230所示的连接子氨基酸序列；VL2包含如SEQ ID NO:115所示的LCDR1氨基酸序列，如SEQ ID NO:133所示的LCDR2氨基酸序列和如SEQ ID NO:142所示的LCDR3氨基酸序列，或分别与所述的LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；CL选自人κ恒定区或人λ恒定区，优选人κ恒定区(如SEQ ID NO:233所示的氨基酸序列)。
在一些具体实施方案中，所述VL1包含如SEQ ID NO:92所示的VL氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述VL2包含如SEQ ID NO:193所示的VL氨基酸序列，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨 基酸序列。
本发明的抗HER2互补性双特异性抗体能够特异性地同时结合HER2胞外区的两个不同表位，因此可以有效交联细胞表面的HER2而形成抗原-抗体交联聚集体或簇，进而诱发细胞内吞并促进内化的簇被转运至溶酶体并在溶酶体内降解。由此，所述互补性双特异性抗体能够显著下调细胞表面HER2的表达量，降低HER2的二聚化，以致抑制HER2过表达的肿瘤细胞的增殖。并且，本发明的抗HER2互补性双特异抗体不干扰HER2介导的信号传导通路，因此其不会干扰正常组织/细胞的HER2的正常生物学功能。
3、抗HER2互补性双特异抗体-药物偶联物(ADC)
本发明进一步提供了抗HER2互补性双特异ADC，所述互补性双特异ADC包含小分子毒素化合物通过可裂解接头偶联至本发明的抗HER2互补性双特异抗体，其具有以下一种或多种功能特性：
(i)所述抗HER2互补性双特异ADC为四价分子，能够特异性地结合肿瘤细胞表面HER2的两个非重叠表位，因此可以有效地交联HER2并在肿瘤细胞膜上形成交联聚集体或簇。一方面，簇的形成导致其被细胞快速且高效地内吞，而且内吞后的胞内转运途径由通常的再循环(Recycling)改变为溶酶体转运(Lysosomal Trafficking)，结果显著提高了所述ADC转运进入溶酶体的效率，从而在溶酶体内降解后能够释放出更多的小分子毒素化合物至细胞质中。因此，所述互补性双特异ADC具有更强的靶向细胞毒性，表现为在HER2过表达的肿瘤细胞(IHC 3+、或IHC 2+/FISH+)中具有比现有的HER2靶向药物(例如，Trastuzumab、Pertuzumab、T-DM1、DS-8201)更强的杀伤活性，并且在HER2低表达的肿瘤细胞(IHC 2+/FISH-、或IHC 1+)中也能发挥直接杀伤作用，具有拓展HER2靶向治疗适应症范围的潜力；另一方面，释放的小分子毒素化合物是疏水性的，具有细胞膜通透性，因此可以被动扩散入肿瘤微环境中发挥旁观者效应(Bystander Effect)，即杀伤邻近的表达更低水平或者不表达HER2的肿瘤细胞。总之，所述互补性双特异ADC相较于普通ADC具有更加广谱的肿瘤细胞杀伤活性，更加难以产生耐药性。
(ii)所述抗HER2互补性双特异ADC对Trastuzumab、Pertuzumab、T-DM1和DS-8201产生耐药性或复发的肿瘤具有杀伤作用，因此能够破解现有HER2靶向药物的耐药性问题。
(iii)所述抗HER2互补性双特异ADC不影响HER2的二聚化及其介导的信号转导通路的调控，因此不会干扰心肌细胞中HER2的正常生物学功能，由此极大地降低了引发心脏毒副作用的可能性。
(iv)所述抗HER2互补性双特异ADC具有显著降低了的Fc受体结合亲和力，因此具有极低的Fc受体介导的安全性风险。
所述ADC可以由式(Ⅰ)表示：
Ab-(L-D)p(Ⅰ)
其中Ab表示本发明所述的抗HER2互补性双特异抗体；
D表示小分子毒素化合物(Drug)；
L表示将Ab偶联至D的可裂解接头(Cleavable Linker)；以及
p表示偶联至Ab上的(L-D)的拷贝数，为2至8。
所述ADC中Ab为本发明的抗HER2互补性双特异抗体，其包含第一和第二抗原结合结构域，其中所述第一抗原结合结构域包含表2所示的抗HER2抗体或其抗原结合片段的VH和VL，能够特异性地结合HER2胞外区的D3，且为Fv结构域；所述第二抗原结合结构域包含表3所示的抗HER2抗体或其抗原结合片段的VH和VL，能够特异性地结合HER2胞外区的D1，且为Fab结构域或IgG结构域。
在一些实施方案中，所述抗HER2互补性双特异ADC的第一抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述VH和VL包含：(i)如SEQ ID NO:11所示的HCDR1；(ii)如SEQ ID NO:24、29、30、或31所示的HCDR2；(iii)如SEQ ID NO:37所示的HCDR3；(iv)如SEQ ID NO:42所示的LCDR1；(v)如SEQ ID NO:46所示的LCDR2；以及(vi)如SEQ ID NO:49所示的LCDR3；或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％相同的氨基酸序列。
在一些实施方案中，所述抗HER2互补性双特异ADC的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述VH和VL包含：(i)如SEQ ID NO:98所示的HCDR1；(ii)如SEQ ID NO:102所示的HCDR2；(iii)如SEQ ID NO:110所示的HCDR3；(iv)如SEQ ID NO:115所示的LCDR1；(v)如SEQ ID NO:133所示的LCDR2；以及(vi)如SEQ ID NO:142所示的LCDR3；或分别与所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施方案中，所述抗HER2互补性双特异ADC的第一抗原结合结构域包含如SEQ ID NO:73、78、79、或80所示的VH，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；和/或如SEQ ID NO:92所示的VL，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
在一些实施方案中，所述抗HER2互补性双特异ADC的第二抗原结合结构域包含如SEQ ID NO:169所示的VH，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；和/或如SEQ ID NO:193所示的VL，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
所述ADC中的D是小分子毒素化合物，当其不缀合于本发明的抗HER2互补性双特异抗体时对肿瘤细胞和正常细胞均具有细胞毒性或细胞生长抑制作用，但所述小分子毒素化合物在缀合形成ADC后，只有当ADC内化和转运入表达HER2的靶细胞溶酶体内，并通过酶促作用、水解、氧化或任何其它机制从所述ADC解离出来后，所述小分子毒素化合物才具有细胞毒性和/或旁观者杀伤效应。所述小分子毒素化合物包括细胞毒素和化疗药物。
在一些实施方案中，所述细胞毒素包括微管蛋白抑制剂和DNA损伤剂，所述微管蛋白抑制剂包括艾日布林(Eribulin)、奥瑞他汀(Auristatins)类衍生物(例如，MMAE、MMAF、MMAD)、Tubulysins、Cryptomycins和美登素(Maytansinoids)类衍生物(例如，DM1、DM2、DM3、DM4)，所述的DNA损伤剂包括拓扑异构酶抑制剂(例如，喜树碱类衍生物SN-38、依沙替康[Exatecan]和DXd)、吡咯并苯并二氮杂卓(PBD)、和卡奇霉素(Calicheamicin)及其衍生物(例如N-乙酰基卡奇霉素[CMC])、倍癌霉素(Duocarmycin)，优选的小分子毒素化合物是艾日布林。
艾日布林的分子结构如式(II)所示：
术语“艾日布林”是指软海绵素B的合成类似物，软海绵素B是从海洋海绵冈田软海绵(Halichondria okadais)中分离出来的大环化合物。艾日布林作为微管动力学抑制剂，通过与微管蛋白结合而抑制有丝分裂纺锤体的形成使得细胞分裂周期停滞于G2/M期。示例性艾日布林或其类似物的结构及其合成的方法描述于WO1999065894、WO2004034990、ZL201910197071.8、和/或ZL201910509222.9中，以引用的方式将其公开内容并入本文中。
在一些实施方式中，小分子毒素化合物为艾日布林，其具有临床治疗窗口大，且脱靶细胞毒性低的特点。
在一些实施方案中，所述化疗药物可以是天然的或合成的化合物，包括但不限于烷基化疗剂以及具有烷基化形式的其他化合物(例如，氮芥、亚乙基亚胺化合物、烷基磺酸盐、亚硝基脲、顺铂、达卡巴嗪)、抗代谢物(例如，叶酸、嘌呤或嘧啶拮抗剂)、有丝分裂抑制剂(例如，长春花生物碱和鬼臼毒素的衍生物)、细胞毒性抗生素(例如，蒽环类抗生物包括柔红霉素和阿霉素，以及放线菌素、博来霉素)。
所述可裂解接头在表达HER2的肿瘤细胞外是稳定的，使得本发明的ADC在体外或血液循环中保持结构稳定，不会因小分子毒素化合物的非靶向或随机释放而导致系统性毒性(脱靶效应)，同时，在内吞进入表达HER2的肿瘤细胞后能够快速进入溶酶体降解并释放出小分子毒素化合物以杀伤肿瘤细胞。所述可裂解接头是指包含可裂解部分的任何接头。如本文所用，术语“可裂解部分”是指可裂解的任何化学键，例如，本领域中众所周知的，包括但不限于，对酸不稳定的键、对蛋白酶/肽酶不稳定的键、对光不稳定的键、对酯酶不稳定的键、以及二硫键。包含可裂解部分的接头可以允许小分子毒素化合物经由接头中的特定位点裂解而从ADC中解离出来。
在一些实施方案中，所述可裂解接头包含可裂解肽部分，所述可裂解肽部分由细胞内蛋白酶(例如，内体[Endosome]蛋白酶、溶酶体蛋白酶或肿瘤相关蛋白酶)裂解后，即可从ADC解离出小分子毒素化合物以杀死肿瘤细胞。在一些实施方式中，所述可裂解肽部分包含氨基酸单元，可以由溶酶体半胱氨酸组织蛋白酶(例如组织蛋白酶B、C、F、H、K、L、O、S、V、X、或W)裂解。所述氨基酸单元可以包含天然存在的氨基酸残基和/或非天然存在的氨基酸类似物，例如瓜氨酸(Citrulline，简称Cit)。在一些实施例中，所述氨基酸单元包含二肽、三肽、或四肽，例如，可以由组织蛋白酶B裂解的Phe-Lys、Val-Cit(VC)、Glu-Val-Cit、或Gly-Gly-Phe-Gly(GGFG)氨基酸序列，优选的氨基酸单元包括VC和GGFG。
在一些实施方案中，所述可裂解接头可以包含至少一种将小分子毒素化合物(D)偶联至本发明的抗HER2互补性双特异抗体(Ab)的间隔区(Spacer)，所述间隔区包含与抗体缀合的间隔区，和/或与小分 子化合物缀合的第二间隔区。
在一些实施方式中，所述与抗体缀合的间隔区可以是亲水性的，以增加ADC的亲水性而改善其稳定性、减少ADC产物聚集和降低免疫原性，示例性的间隔区包含一个或多个聚乙二醇(PEG)，例如1个、2个、3个、4个、5个、或6个PEG，优选2个PEG。在一些实施例中，所述间隔区经由顺丁烯二酰亚胺(Mal)附接于抗体，其中，经由Mal附接于抗体的间隔区在本文中又可称为“Mal-间隔区”。在一些实施例中，所述Mal-间隔区包含一个或多个PEG部分(例如2个PEG)。
在一些实施方式中，所述与小分子化合物缀合的第二间隔区用于将可裂解接头的可裂解部分(例如可裂解肽)附接于小分子化合物。在一些实施例中，所述与小分子化合物缀合的第二间隔区具有自焚碎裂(Self-immolation)的特性，有利于小分子化合物在靶细胞内被完整释放，即在靶细胞内释放出的小分子化合物不带有与其缀合的间隔区部分或其它修饰性基团，使得释放的小分子化合物不因带有修饰性基团而影响其抗肿瘤活性。在一些实施例中，所述自焚碎裂间隔区包含对氨基苯甲基单元，对氨基苯甲醇(PABOH)经由酰胺键缀合于可裂解接头的可裂解部分(例如，可裂解肽的氨基酸单元)，且在PABOH与小分子化合物之间形成氨基甲酸酯基、甲基氨基甲酸酯基或碳酸酯基(Hamann等,Expert Opinion on Therapeutic Patents 2005,15:1087-1103)。进一步，所述自焚碎裂间隔区包含式(Ⅲ)所示的对氨基苯甲基羰基(PAB)，PAB的自焚碎裂涉及自发的1,6-消去反应(Jain等,Pharm Res 2015,32:3526-3540)：
在一些实施方案中，所述可裂解接头包含Mal-间隔区和可裂解肽部分。在一些实施方式中，所述间隔区包含PEG部分(例如2个PEG)，所述可裂解肽部分包含氨基酸单元(例如，二肽VC和四肽GGFG)。在一些实施方案中，所述可裂解接头包含共价连接的Mal-间隔区-氨基酸单元，其中，所述氨基酸单元为Phe-Lys、VC、Glu-Val-Cit或GGFG。在一些实施方案中，所述可裂解接头包含共价连接的Mal-间隔区-氨基酸单元-PAB，其中，所述氨基酸单元为Phe-Lys、VC、Glu-Val-Cit、或GGFG。在一些实施例中，所述间隔区为(PEG)m，m为1-5的整数，优选2。在一些实施例中，所述可裂解接头包含Mal-(PEG)2和VC。在一些实施例中，所述可裂解接头包含Mal-(PEG)2和GGFG。
在一些实施方式中，所述可裂解接头包含结构：Mal-间隔区-可裂解肽部分。在一些实施例中，所述可裂解接头包含结构：Mal-(PEG)2-VC和Mal-(PEG)2-GGFG。
在一些实施方案中，所述可裂解接头包含Mal-间隔区、可裂解肽部分和第二间隔区。在一些实施例中，所述间隔区包含PEG部分(例如2个PEG)，所述可裂解肽部分包含氨基酸单元(例如，二肽VC)，所述第二间隔区是自焚碎裂的(例如，PAB)。在一些实施例中，所述可裂解接头包含Mal-(PEG)2、VC和PAB。
在一些实施方式中，所述可裂解接头包含结构：Mal-间隔区-可裂解肽部分-第二间隔区。在一些实施例中，所述可裂解接头包含结构：Mal-(PEG)2-VC-PAB。
在一些实施方案中，所述可裂解接头的Mal-间隔区与本发明ADC中的抗体部分的一个或多个氨基酸残基偶联，例如，Mal-间隔区可通过硫醇基团偶联至抗体，包括偶联至抗体的一个或多个半胱氨酸的巯基。在一些实施方式中，所述Mal-间隔区的顺丁烯二酰亚胺基与所述抗体的半胱氨酸残基的巯基反应，从而使Mal-间隔区与抗体缀合。在一些实施方案中，所述Mal-间隔区的顺丁烯二酰亚胺基可以与所述抗体的恒定区和/或可变区中特定位置处的半胱氨酸残基的巯基反应，进一步，所述Mal-间隔区的顺丁烯二酰亚胺基可以与所述抗体铰链区的二硫键和/或链间二硫键经还原后释放出来的游离半胱氨酸残基偶联。在一些实施例中，所述Mal-间隔区的顺丁烯二酰亚胺基与所述抗体铰链区的半胱氨酸残基偶联，可以将抗体铰链区的链间二硫键通过化学反应(例如，还原、pH调节或水解)产生多个游离的半胱氨酸残基，也可以将抗体恒定区的一个或多个特定位点的氨基酸残基通过DNA重组技术(例如通过以半胱氨酸残基取代或插入的方式)而产生工程化半胱氨酸残基。
在一些实施方案中，所述可裂解接头的第二间隔区与小分子化合物偶联，所述小分子化合物是艾日布林或其衍生物。在一些实施方式中，所述第二间隔区将可裂解接头中的可裂解肽部分偶联至艾日布林的C-35胺，所述第二间隔区是自焚碎裂的(例如，PAB)，所述可裂解肽部分包含VC，自焚碎裂间隔区在可裂解肽部分经酶解后自发进行自焚碎裂，导致艾日布林以天然活性形式从ADC中释放。在一些实施例中，自焚碎裂间隔区是PAB，将可裂解肽部分VC缀合于艾日布林的C-35胺，以构建为包含Mal-(PEG)2-VC-PAB-Eribulin结构的ADC。
在一些实施方案中，所述可裂解接头的可裂解部分(例如可裂解多肽)可以直接缀合于所述ADC的小分子毒素化合物部分，所述小分子毒素化合物是艾日布林或其衍生物。在一些实施方式中，所述可裂解 多肽包括氨基酸单元，所述氨基酸单元包含GGFG，该四肽通过羧基与艾日布林的C-35胺缀合，以构建为包含Mal-(PEG)2-GGFG-Eribulin结构的ADC。采用可裂解接头中的GGFG四肽直接与艾日布林缀合的方式，同样可以保证所述ADC的可裂解接头在靶细胞内经裂解后能够完整地释放出具有天然活性形式的艾日布林，使其发挥靶向杀伤肿瘤细胞的作用和/或旁观者效应。
在一些实施方案中，本发明的ADC结构包括本发明所述的抗HER2互补性双特异抗体通过可裂解接头缀合于艾日布林或其衍生物，所述可裂解接头包含Mal-间隔区、可裂解肽部分和/或第二间隔区。在一些实施方式中，所述ADC结构包含本发明的抗HER2互补性双特异抗体通过可裂解接头与艾日布林的C-35胺缀合，所述可裂解接头包含Mal-(PEG)2-VC-PAB或Mal-(PEG)2-GGFG，所述抗体铰链区的二硫键和/或链间二硫键经还原后释放出来的游离半胱氨酸残基与所述可裂解接头的Mal缀合。
在一些实施方式中，本发明所述的ADC包含如下部分：
在一些实施方案中，本发明的ADC包括本发明的抗HER2互补性双特异抗体与药物有效载荷(Payload)偶联，所述药物有效载荷包括Mal-(PEG)2-VC-PAB-Eribulin和Mal-(PEG)2-GGFG-Eribulin。在一些实施方式中，所述ADC包含本发明的抗HER2互补性双特异抗体通过其半胱氨酸残基与药物有效载荷的Mal缀合，进一步，所述互补性双特异抗体铰链区的二硫键和/或链间二硫键经还原后释放出来的游离半胱氨酸残基与所述药物有效载荷的Mal缀合。
在一些实施方案中，本发明提供了具有下式的ADC：

其中，Ab表示本发明所述的抗HER2互补性双特异抗体，p为2至8。
所述式(Ⅰ)中的p在本文中也称为药物与抗体比率(Drug-to-Antibody Ratio，简称DAR)或有效载荷分子的数量，即抗体所偶联的小分子化合物的个数，所述p为2至8，例如，4至8。本发明所述的ADC所包含的抗HER2互补性双特异抗体通过可裂解接头与艾日布林缀合的DAR值具有合乎需要的特性，即所述ADC不仅具有足够的杀伤活性，而且具有足够的稳定性。一般而言，较高的DAR值(例如，p＞8)可能会因为小分子毒素化合物的强疏水性而使得ADC在水相环境中因疏水作用而发生聚集和沉淀，甚至会在体内引发安全性风险；较低的DAR值(例如，p＜2)可能会导致ADC对肿瘤细胞的杀伤活性较低而影响疗效。在一些实施方案中，最佳DAR值约为4至8。本发明的抗HER2互补性双特异ADC的平均DAR值(即平均有效载荷分子数或平均p)可以利用本领域中已知的常规分析方法(例如，反相LC-MS质谱分析和/或HIC-HPLC)的结果通过计算来获得。
4、制备本发明所述抗体的方法
4.1多核苷酸、载体和宿主细胞
一方面，本发明提供了一种编码抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的核酸。本发明还包括编码本文所述氨基酸序列的多核苷酸变异体。
对应于本发明所述的氨基酸序列、用作核酸分离的探针或引物或者数据库提供可查询的核苷酸序列可以通过氨基酸序列回译来获得。聚合酶链反应(PCR)程序可以用以分离和扩增编码本发明的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体的DNA序列。界定DNA片段组合的所需末端的寡核苷酸用作5'和3'引物。寡核苷酸可以另外含有限制内切核酸酶的识别位点，以促进扩增DNA片段组合插入表达载体中。PCR技术参见于Saiki等,Science 1988,239:487-491；Wu等编著,Recombinant DNA Methodology,1989 Academic Press,第189-196页；Innis等编著,PCR Protocols:A Guide to Methods and Applications,1990 Academic Press。
本发明的核酸分子包括单股和双股形式DNA和RNA，以及对应的互补序列，包括分离的核酸分子，优选来源于至少一次呈基本上纯的形式分离并且数量或浓度能够通过标准生物化学法鉴别、操作和回收其组分核苷酸序列的DNA或RNA(例如Sambrook等,Molecular Cloning:A Laboratory Manual,1989第2版，Cold Spring Harbor Laboratory中所述的方法)。优选地，此类序列包括以不常存在于真核基因中的内部非翻译序列或内含子中断的开放阅读框的形式所提供和/或构建。非翻译DNA的序列可以存在于开放阅读框的5'或3'，其中所述序列不干扰编码区的操控或表达。
本发明的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体通过以下步骤来制备：使用PCR诱变或本领域一般技术人员已知的其它技术对编码抗HER2抗体或其抗原结合片段或抗HER2互补性双特异抗体的DNA中的核苷酸进行位点特异性诱变以产生编码变异体的DNA，此后在如本文中概述的细胞培养物中表达重组DNA。此外，抗HER2抗体或其抗原结合片段和抗HER2互补性双特异抗体也可以通过使用已建立的技术体外合成来制备。
如本领域技术人员所已知的，由于遗传密码的简并，本发明的抗HER2抗体或其抗原结合片段或抗HER2互补性双特异抗体由极其大量的核酸进行编码，每个部分的核酸都在本发明的范围内并且可以使用标准技术制成。因此，鉴别了特定的氨基酸序列，本领域的技术人员可以通过以不改变本发明的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体氨基酸序列的方式来简单对其各自的编码序列进行一个或多个以上密码子修饰以制备许多不同的核酸。
另一方面，本发明还提供了编码本文的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的核酸的表达载体。
可以将编码本发明的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的核酸构建在合适的载体中，以导入宿主细胞进行目的蛋白的表达。载体组分通常包括但不限于以下一种或多种：信号序列、复制起点、一种或多种标记基因、增强子元件、启动子和转录终止序列。载体中编码目标蛋白的核酸与启动子可操作地连接。
如本文所使用的，术语“可操作地连接”是指核酸表达控制序列(例如，启动子、信号序列或转录调节因子结合位点的阵列)与另一个核酸序列之间的功能性连接，并因此该控制序列控制其他核酸序列的转 录和/或翻译。
合适的载体包括质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬菌体或M13噬菌体，以及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于病毒颗粒、脂质体或蛋白外壳。
另一方面，本发明还提供了包含编码本发明的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的核酸或表达载体的宿主细胞。
该细胞可以是真核细胞，例如，哺乳动物宿主细胞，包括但不限于，SV40转化的猴肾细胞CV1系(COS-7,ATCC,CRL-1651)、人胚胎肾细胞系(293或悬浮培养的293细胞亚克隆，Graham等,J Gen Virol 1977,36:59-74)、幼地鼠肾细胞(BHK-21，ATCC,CCL-10)、中国仓鼠卵巢细胞/-DHFR(CHO,Urlaub等,Proc Natl Acad Sci USA 1980,77:4216-4220)、小鼠睾丸支持细胞(TM4,Mather,Biol Reprod 1980,23:243-251)、猴肾细胞(CV1,ATCC,CCL-70)、非洲绿猴肾细胞(VERO-76,ATCC,CRL-1587)、人宫颈癌细胞(HELA,ATCC,CCL-2)、犬肾细胞(MDCK,ATCC,CCL-34)、布法罗大鼠肝细胞(BRL 3A，ATCC,CRL-1442)、人肺细胞(W138,ATCC,CCL-75)、人肝癌细胞系(HepG2,ATCC,HB-8065)、小鼠乳腺瘤(MMT060562,ATCC,CCL-51)、TRI细胞(Mather等，Ann NY Acad Sci 1982,383:44-68)、MRC5细胞、或FS4细胞。4.2本发明的抗HER2抗体的筛选
本发明提供的抗HER2抗体可以是鼠源抗体，也可以是嵌合抗体、人源化抗体或全人源抗体。产生单克隆抗体的方法是本领域已知的，任何一种已知的方法(例如，杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等)均可用于本发明中，以制备特异性结合HER2的单克隆抗体。
在一些实施方式中，可选择HER2氨基酸序列中任意片段的多肽(优选HER2胞外区)作为目的抗原免疫小鼠，并从小鼠成熟B细胞中扩增获得抗体可变区基因，通过噬菌体展示技术将小鼠抗体可变区展示在噬菌体表面，构建噬菌体展示文库，并通过特定靶分子(例如，用于免疫小鼠的目的抗原)进行淘选，检测噬菌体表面所展示的抗体可变区与所述靶抗原的相互作用，通过体外选择的方法筛选并扩增抗体可变区文库，所述体外选择类似于天然选择。
在一些实施方案中，本发明通过构建噬菌体展示文库并针对目的抗原进行淘选(Panning)，从抗体文库中筛选出识别目的抗原的抗体。可利用多种本领域已知的噬菌体展示方法来产生本发明的抗体，例如，US5223409、US5622699和US6068829公开了用于制备噬菌体文库的方法。也可根据“Antibody Phage Display:Methods and Protocols(O′Brien和Aitken编辑)”中所描述的方法来构建噬菌体展示文库。在一些实施方式中，可以将编码抗体可变区的核酸插入至噬菌体外壳蛋白基因中，使得噬菌体将该抗体可变区展示在其表面，同时，在噬菌体内部含有编码该抗体可变区的核酸，从而实现抗体可变区表型和基因型之间的联系。
关于抗体(或单链抗体)的筛选，在噬菌体展示中，抗体(或单链抗体)的VH和/或VL区的较大文库可在丝状噬菌体颗粒的表面上表达，由此使其配对形成结合域。可根据与目的抗原识别和结合及其所展示的结合域，从文库中筛选出噬菌体。
在一些实施方式中，所述淘选可以通过噬菌体感染宿主细菌并在所述宿主中繁殖扩增来实现。由宿主细菌分泌的表面展示有单链抗体片段的噬菌体经收集后，可按需要进行多轮次的淘选直至得到能够选择性或特异性结合靶抗原的噬菌体，最后通过对噬菌体基因组中的抗体基因进行测序来获得抗体可变区的氨基酸序列(Arap等,Science 1998,279:377-380；Smith等,Science 1985,228:1315-1317)。
在特定的实施方式中，本发明利用HER2胞外区重组蛋白作为目的抗原免疫小鼠，从经免疫的小鼠脾脏组织中得到抗体重链可变区族和轻链可变区族基因片段，基于此制得scFv片段，并连接在噬菌体表面结构蛋白基因III上，利用共表达方式，使之参与噬菌体组装并展示在噬菌体表面，由此构建获得噬菌体展示文库。利用噬菌体展示文库，针对特定靶抗原(例如，用于免疫小鼠的抗原)，通过多轮吸附-洗脱-扩增的重复过程(淘选)，可富集能特异性结合靶抗原的噬菌体，再通过基因测序技术得到相应的DNA序列信息，进而推断抗体可变区的氨基酸序列。
5、制备方法
5.1抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的制备方法
本发明提供了使用所述宿主细胞制备本发明所述的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的方法。
所述方法包括将编码本发明的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体的核酸或表达载体转染到宿主细胞中，并在培养基中培养宿主细胞一段时间以表达本发明的抗HER2抗体或其抗 原结合片段、或抗HER2互补性双特异抗体。不受限制地，商业上可获得的培养基可以用作细胞培养基。
优选地，表达的抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体可分泌到培养宿主细胞生长的培养基中。使用常规蛋白质纯化的方法从培养基中回收抗体，例如通过离心或超滤除去杂质，或通过亲和色谱法纯化所得物；还可以使用其他纯化技术，例如阴离子或阳离子交换色谱法、疏水相互作用色谱法和羟基磷灰石色谱法。
5.2抗HER2互补性双特异ADC的制备方法
本发明的ADC可通过本领域已知的任何方法制备得到，所述方法包括但不限于：(1)抗体的亲核基团或亲电子基团与可裂解接头反应，经由共价键形成抗体-可裂解接头中间产物(Ab-L)，再与小分子毒素化合物(D)的反应，所述中间产物Ab-L在与小分子毒素化合物反应之前可以经由纯化步骤，也可以不经由纯化步骤；(2)小分子毒素化合物的亲核基团或亲电子基团与可裂解接头反应，经由共价键形成小分子毒素化合物-可裂解接头中间产物(D-L)，再与抗体的亲核基团或亲电子基团的反应，所述中间产物D-L在与抗体反应之前可以经由纯化步骤，也可以不经由纯化步骤；或(3)可以将抗体、可裂解接头和小分子毒素化合物混合反应，使得抗体与可裂解接头之间以及可裂解接头与小分子毒素化合物之间同时形成共价键，制备为本发明的ADC。在本领域中己知制备ADC的方法的几个具体实例，如US8624003(一锅法)、US8163888(一步法)和US5208020(两步法)所描述的。
在一些实施方案中，所述抗体在进行缀合反应前置于还原条件，以产生一个或多个游离的半胱氨酸残基。在一些实施方式中，使用还原剂(例如，二硫苏糖醇[DDT]、2-巯基乙醇或三(2-羧乙基)膦[TCEP])与抗体作用还原抗体铰链区的链间二硫键，以产生部分或完全还原的具有游离巯基的抗体。所述还原剂优先还原抗体铰链区的链间二硫键，而抗体的链内二硫键保持完整。在一些实施例中，在含有螯合剂的缓冲液中，使用还原剂TCEP与抗体反应，可得到部分或完全还原的具有游离巯基的抗体。所述螯合剂包括但不限于乙二胺四乙酸(EDTA)、二亚乙基三胺五乙酸(DTPA)，所述缓冲液包括但不限于组氨酸-盐酸、磷酸钠、硼酸钠、乙酸钠溶液。
在一些实施方案中，上述得到的部分或完全还原的具有游离巯基的抗体可以与可裂解接头(L)或小分子毒素化合物-可裂解接头中间产物(D-L)的反应官能团(例如，顺丁烯二酰亚胺基)反应形成共价键(例如，硫醚键)，以得到抗体-可裂解接头中间产物(Ab-L)或ADC。在本文中，所述的小分子毒素化合物-可裂解接头中间产物(D-L)与所述的药物有效载荷(Payload)互换使用。
上述方法制备得到的ADC的纯化方法可以是本领域中已知的用于纯化蛋白质的任何生物化学方法或其任何方法组合，包括但不限于亲和色谱法、离子交换色谱法、混合模式色谱法(例如，陶瓷羟基磷灰石层析)、疏水作用色谱法、空间排阻色谱法、渗析法、过滤法、选择性沉淀法或其任何组合。
6、药物组合物
本发明提供了药物组合物，该组合物包含本发明所述的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC，以及药学上可接受的载体。
所述药物组合物可以包含任何种类的药学上可接受的载体。可以使用的载体包括赋形剂、表面活性剂、增稠剂或乳化剂、固体粘合剂、分散或悬浮助剂、增溶剂、着色剂、调味剂、包衣剂、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂或其组合。例如，以下著作教导了选择和使用合适的载体：Gennaro编著,Remington:The Science and Practice of Pharmacy,2003第20版(Lippincott Williams&Wilkins),以引用的方式将其公开内容并入本文。
优选地，药物组合物适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮给药。如本发明所使用的“肠胃外给药”是指非肠内和局部的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、包膜下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。或者，本发明的抗体还可以通过非肠胃外途径(例如，局部、表皮或粘膜给予途径)给药，例如，鼻内、口服、阴道、直肠、舌下或局部给药。鉴于不同的给药途径，可以将活性成分包被在材料中以保护其免受酸/碱和可能使其失活的其它自然条件的作用。
药物组合物可以是无菌水性溶液或分散剂。它们也能够形成微乳液、脂质体、或其它适合于高药物浓度的有序结构组合形式。
可以与载体材料组合以形成单一剂型的活性成分的量是依据受试者和特定的给药方式所确定的，并且通常也是能够产生治疗效果的组合物的量。通常，与药学上可接受的载体形成的组合物包含约0.01％至约99％的活性成分，优选约0.1％至约70％、最优选约1％至约30％的活性成分。
调整剂量方案以提供最佳的预期应答(例如治疗应答)。例如，可以单次给药，可以分数次给药，或者可以根据治疗情况按比例减少或增加剂量。以剂量单位形式配制肠胃外给药的组合物是特别有利的，使给药方便和给药剂量均匀。如本发明所使用的，剂量单位形式是指物理上离散的单位，其适合作为治疗受试者的单位剂量；每个单位剂量包含预定量的活性成分，所述活性成分的预定量是通过计算活性成分与所 需的药物载体一起施用产生预期治疗效果而得到的。另外，本发明的抗HER2抗体、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC也可以作为缓释制剂给药，这样可减少给药频率。
本发明的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC，或包含抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC的组合物给药剂量的范围可为约0.0001mg/kg至100mg/kg体重，通常为0.001mg/kg至50mg/kg体重。
本发明的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC的“治疗有效剂量”优选可使得疾病症状严重性降低、疾病症状无进展期的频率和持续时间增加、或预防由疾病困扰所造成的机体损伤或失能。例如，相对于未治疗的受试者，荷瘤受试者的“治疗有效剂量”优选抑制肿瘤生长至少约20％、更优选至少约40％、甚至更优选至少约60％、还可以更优选至少约80％。治疗性抗体或ADC的治疗有效量可以减少肿瘤的大小、或改善受试者的症状，所述受试者通常是人或其它哺乳动物。“治疗有效剂量”还可以根据各种因素而不同地确定，所述的各种因素包括但不限于配制方法、给药方法、年龄、身体、体重、患者的性别或病理状况、饮食、给药时间、给药间隔、给药途径、排泄率和反应敏感性。
药物组合物可以选择控释制剂，包括植入物、透皮贴剂和微囊化的递送系统。可以使用可生物降解的生物相容性聚合物，例如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯和聚乳酸。参见例如，Robinson编著,Sustained and Controlled Release Drug Delivery Systems,1978(Marcel Dekker)。
可以通过如下的医疗装置来传输治疗性药物组合物，所述医疗装置选自：(1)无针皮下注射装置(例如，US5399163、US5383851、US5312335、US5064413、US4941880、US4790824和US4596556)；(2)微型输注泵(US4487603)；(3)透皮装置(US4486194)；(4)输注设备(US4447233和US4447224)；以及(5)渗透装置(US4439196和US4475196)；以引用的方式将其公开内容并入本文。
在某些实施方案中，可以配制本发明的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或抗HER2互补性双特异ADC以确保在体内的生物分布。例如，为了确保本发明的治疗性抗体或ADC穿过血脑屏障，可以将其配制成脂质体，所述脂质体可以另外包含靶向部分，以增强向特定细胞或器官的选择性递送。参见例如，US4522811、US5374548、US5416016和US5399331；Ranade,J Clin Pharmacol 1989,29:685-694；Umezawa等,Biochem Biophys Res Commun 1988,153:1038-1044；Bloeman等,FEBS Lett 1995,357:140-144；Owais等,Antimicrob Agents Chemother 1995,39:180-184；Briscoe等,Am J Physiol 1995,268:L374-380；Schreier等,J Biol Chem 1994,269:9090-9098；Keinanen和Laukkanen,FEBS Lett 1994,346:123-126；以及Killion和Fidler,Immunomethods 1994,4:273-279。
7、药盒
本发明提供了一种药盒，其包含有效量的本发明的抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、或药物组合物，以及任选的至少一种另外的肿瘤治疗剂(即，所述药盒可包含或不含至少一种另外的肿瘤治疗剂)。优选地，所述肿瘤治疗剂可包括但不限于ErbB2/HER2的另一种拮抗剂；EGFR拮抗剂、HER3拮抗剂；MET拮抗剂，MET的小分子抑制剂(例如Capmatinib)；IGF1R拮抗剂(例如，抗IGF1R抗体)；B-Raf抑制剂(例如，维罗非尼、索拉非尼、GDC-0879、PLX-4720)；PDGFR-α抑制剂(例如，抗PDGFR-α抗体)；PDGFR-β抑制剂(例如，抗PDGFR-β抗体或小分子激酶抑制剂[例如，甲磺酸伊马替尼或苹果酸舒尼替尼])；PDGF配体抑制剂(例如，抗PDGF-A抗体、抗PDGF-B抗体、抗PDGF-C抗体或抗PDGF-D抗体、适体、siRNA等)；VEGF拮抗剂(例如，VEGF-Trap，比如阿柏西普)；抗VEGF抗体(例如，贝伐单抗)；VEGF受体激酶抑制剂(例如，舒尼替尼、索拉非尼或帕唑帕尼)；DLL4拮抗剂(例如，REGN421)；Ang2拮抗剂(例如，WO2011014469公开的抗Ang2抗体，比如H1H685P)；FOLH1拮抗剂(例如，抗FOLH1抗体)；STEAP1或STEAP2拮抗剂；TMPRSS2拮抗剂；MSLN拮抗剂；MUC16拮抗剂；CLEC12A拮抗剂；PD-1或PD-L1阻断剂(例如，Pembrolizumab或Nivolumab)；激素受体调节剂(例如，雌激素受体调节剂[比如他莫昔芬]；雄性激素受体调节剂)；芳香酶抑制剂(例如，来曲唑、阿那曲唑、依西美坦)；激酶抑制剂(例如，酪氨酸激酶抑制剂[比如拉帕替尼])；细胞因子激动剂；细胞因子抑制剂(包括小分子细胞因子抑制剂和与细胞因子诸如IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-8、IL-9、IL-11、IL-12、IL-13、IL-17、IL-18或其各自的受体结合的抗体)；化学治疗剂(包括但不限于微管破坏物、抗代谢物、拓扑异构酶抑制剂、DNA嵌入剂、烷化剂)等。
在一些实施方案中，所述ErbB2/HER2的另一种拮抗剂包括抗HER2单克隆抗体(例如，Trastuzumab、Pertuzumab)和/或ADC(例如，T-DM1、DS-8201)，或抗ErbB2/HER2小分子抑制剂(例如，酪氨酸激酶抑制剂类的拉帕替尼、吡咯替尼和奈拉替尼等)；所述EGFR拮抗剂包括抗EGFR抗体(例如，西妥昔单抗、帕尼单抗)、抗EGFR小分子抑制剂(例如，Gefitinib、Erlotinib)、抗EGFRvIII拮抗剂(例如，抗EGFRvIII抗体)；所述HER3拮抗剂包括但不限于抗HER3抗体(例如，Patritumab)；所述MET拮 抗剂包括但不限于抗MET抗体(例如，奥那妥珠单抗、依玛妥珠单抗和H4H14639D)。
8、治疗表达HER2的癌症的方法和用途
一方面，本发明涉及一种治疗表达HER2的癌症的方法，所述方法包括向有需要的受试者施用有效量的本发明所述的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、或发明药物组合物或药盒。或者，本发明涉及上述抗体、ADC、药物组合物或药盒在用于制备对表达HER2的癌症进行治疗的药物中的用途。或者，本申请涉及用于对表达HER2的癌症进行治疗的上述抗体、ADC、药物组合物或药盒。
所述癌症包括但不限于乳腺癌、卵巢癌、宫颈癌、结直肠癌、胃癌、食管癌、肺癌、头颈癌、黑色素瘤、胰腺癌、肝癌、胆管癌、肾癌、膀胱癌、甲状腺癌、前列腺癌、子宫内膜癌，所述癌症还包括各阶段的癌症，例如早期癌症、非转移性癌症、原发性癌症、晚期癌症、局部晚期癌症、转移性癌症或缓解中的癌症。所述癌症可以是HER2过表达或低表达的。所述的受试者可为人、非人灵长类动物或其它哺乳动物如狗、小鼠、大鼠。
如本领域已知的，表达HER2的癌症可以通过癌细胞表面的HER2表达量(即通过“HER2状态”)来表征。HER2的表达量可通过免疫组织化学法(IHC)、荧光原位杂交法(FISH)等方法进行评估。在一些实施方案中，所述癌症包括HER2过表达的癌症(例如，IHC 3+、或IHC 2+/FISH+)、和/或HER2低表达的癌症(IHC 2+/FISH-、或IHC 1+)。
在一些实施方案中，所述抗HER2互补性双特异ADC或其药物组合物对HER2过表达的肿瘤/癌症和/或HER2低表达的肿瘤/癌症均具有杀伤作用。
在一些实施方案中，所述抗HER2互补性双特异抗体或其药物组合物对HER2过表达的肿瘤细胞的生长或增殖具有抑制活性。
在一些实施方案中，向有需要的受试者施用有效量的本发明的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、药物组合物、或药盒可以缩小所述受试者肿瘤体积、抑制肿瘤生长、延长受试者的无病生存期或无进展生存期、增加受试者的总生存期、减少肿瘤转移、或改善受试者生活质量。
另一方面，本发明还涉及一种治疗对现有HER2靶向治疗剂产生耐药性或复发的癌症的方法，所述方法包括向有需要的患者施用有效量的本发明的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、药物组合物、或药盒。或者，本发明涉及所述抗体、ADC、药物组合物、或药盒在用于制备对HER2靶向治疗剂产生耐药性或复发的癌症进行治疗的药物中的用途。
在一些实施方案中，所述患者对一种或多种现有的HER2靶向治疗剂不响应或响应较差，所述HER2靶向治疗剂包括Trastuzumab、Pertuzumab、T-DM1、DS-8201。所述不响应表现为患者的肿瘤生长、肿瘤体积增加、肿瘤转移形成或转移数目增加，不响应还可以是肿瘤转移发展或疾病进展时间缩短。所述响应较差是指患者在经历HER2靶向治疗剂的标准疗法过程中或之后的短时间内其肿瘤生长或转移。
再一方面，本发明还涉及一种治疗对不适合接受现有HER2靶向疗法或难治疗、或在接受现有HER2靶向疗法后产生耐药性或复发的患者的方法，所述方法包括向有需要的患者施用有效量的本发明的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、药物组合物、或药盒。或者，本发明涉及所述抗体、ADC、药物组合物、或药盒在用于制备对不适合接受现有HER2靶向疗法或难治疗、或在接受现有HER2靶向疗法后产生耐药性或复发的患者进行治疗的药物中的用途。所述的HER2靶向疗法包括使用Trastuzumab、Pertuzumab、T-DM1、或DS-8201进行治疗。
在一些实施方案中，当患者对现有的HER2靶向疗法已出现疾病进展时，本发明所述的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、药物组合物、或药盒可用于治疗进展性癌症。
再一方面，本发明的抗HER2互补性双特异抗体、抗HER2互补性双特异ADC、药物组合物、或药盒可单独使用，也可与本领域已知的其它类型的癌症疗法，例如，外科手术、化学疗法、放射疗法、基因疗法、免疫疗法、光动力疗法、射频消融等组合施用。
9、检测用途
本发明还涉及所述抗HER2抗体或其抗原结合片段或抗HER2互补性双特异抗体用于检测和/或测量样品中的HER2或表达HER2的肿瘤细胞的用途，以及用于筛选对本发明的抗HER2互补性双特异ADC治疗有响应的癌症患者的用途。或者，本申请涉及一种检测和/或测量样品中的HER2或表达HER2的肿瘤细胞的方法，以及筛选对上述ADC治疗有响应的癌症患者的方法，包括将所述抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体与所述样品或分离自所述患者的生物样本进行孵育，检测所述抗体是否结合至所述样品或生物样本。或者，本申请涉及用于检测和/或测量样品中的HER2或表达HER2的肿瘤细胞、或筛选对上述ADC治疗有响应的癌症患者的上述抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体。
在一些实施方案中，所述抗HER2抗体或其抗原结合片段、或抗HER2互补性双特异抗体可以用于诊断HER2异常表达(例如，过表达、表达不足或表达缺乏)的病症或疾病，以利于确定治疗方案。例如， 可以将所述抗体与可检测标记物或报告分子偶联标记，并将所标记的抗体与从患者处获得的样品接触以诊断测定HER2表达情况。所述可检测标记物或报告分子可以是放射性同位素，例如3H、14C、32P、35S、或125I；荧光材料，例如，伞形酮、萤光素、罗丹明、异硫氰酸荧光素、二氯三嗪胺荧光素、丹磺酰氯或藻红蛋白；化学发光材料，例如鲁米诺；生物发光材料，例如，荧光素酶、荧光素或水母发光蛋白；或酶，例如，碱性磷酸酶、β-半乳糖苷酶、乙酰胆碱酯酶、辣根过氧化物酶或萤光素酶。可以用于检测或测量样品中的HER2的具体示例性测定法包括酶联免疫吸附测定法(ELISA)、放射免疫测定法(RIA)、免疫PET(例如，89Zr、64Cu等)和荧光活化细胞流式分析法(FACS)。
通过以下的实施例进一步阐释了本发明，其不应被解释为进一步限制。本发明全文中引用的所有附图和所有参考文献、专利和专利申请的内容以引用的方式明确地并入本文。除非另有说明，以下的实施例中涉及的材料、试剂和装置为可商购的。
实施例
实施例1.抗HER2免疫文库的构建以及抗体的筛选
1.1抗HER2免疫文库的构建
利用人HER2胞外区重组蛋白(序列源自NP_004439.2的第23-652位)免疫2只BALB/c小鼠。小鼠经过3次免疫后，采血并利用ELISA检测免疫效价，结果显示免疫效价均达到1:1,000,000以上(数据未显示)。对小鼠进行加强免疫，3天后于无菌条件下摘取小鼠脾脏，并制备小鼠脾脏单细胞悬浮液。利用Animal Total RNA Isolation Kit(FuGene)将收集的小鼠脾脏细胞裂解后进行RNA提取；以提取的RNA为模板，利用逆转录试剂盒(PrimeScriptTMII First Strand cDNA Synthesis Kit,Takara)进行逆转录反应而获得cDNA；随后以cDNA为模板，利用特异引物通过PCR方法分别扩增获得全套小鼠抗体重链可变区族和轻链可变区族的基因片段，再通过重叠PCR(SOE-PCR)将轻、重链可变区基因片段各自拼接扩增而得到单链抗体可变区基因片段(scFv)，将scFv序列通过酶切连接至噬菌粒展示载体中，然后将连接产物电转至TG1感受态细胞中，菌液经过辅助噬菌体M13KO7侵染后获得2个噬菌体展示文库。
1.2抗HER2抗体的筛选与序列分析
以HER2胞外区重组蛋白为靶抗原分别对本实施例第1.1项所述的2个噬菌体展示文库进行淘选，经过3～5轮的“吸附-洗脱-扩增”淘选，再通过单克隆ELISA筛选和PCR酶切实验验证，以及对候选克隆进行测序分析，最终获得15个序列特异的抗HER2鼠源单克隆抗体。
1.3抗HER2嵌合抗体的制备
将上述筛选获得的15个鼠源抗体的轻、重链基因分别与线性化的表达载体pcDNA3.1进行同源重组(重链可变区[VH]与含人源IgG1恒定区的pcDNA3.1载体连接，轻链可变区[VL]与含人源CK恒定区的pcDNA3.1载体连接)，通过菌落PCR检测以及DNA测序分析获得序列正确的嵌合抗体表达载体。利用常规方法提取并纯化获得嵌合抗体表达载体，然后将15组含轻、重链基因片段的嵌合抗体表达载体分别瞬转到ExpiCHO-S细胞(Thermo)中，通过无血清培养进行嵌合抗体的表达，利用AKTA Pure系统进行Protein A亲和层析纯化获得抗HER2嵌合抗体蛋白。
1.4抗HER2嵌合抗体与ErbB/HER家族成员的结合活性检测
利用ELISA方法检测抗HER2嵌合抗体与人源ErbB/HER家族成员的结合活性。具体方法如下：用PBS稀释HER2胞外区重组蛋白以及人源ErbB/HER家族其它成员(EGFR、HER3和HER4)的胞外区重组蛋白，然后以100μL/孔加入到96孔ELISA板中，4℃孵育过夜。PBS洗板后每孔加入200μL封闭液(含有1％ BSA的PBST[PBS+0.1％ Tween-20])，室温静置孵育1小时。PBST洗板后以每孔100μL加入梯度稀释于封闭液中的待测抗体(包括抗HER2嵌合抗体和对照抗体Trastuzumab、Cetuximab或Patritumab)，室温静置孵育2小时。PBST洗板后以每孔100μL加入1:10,000稀释于封闭液中的抗-人IgG(H+L)-HRP抗体(Jackson Immuno)，室温静置孵育1小时。PBST洗板后每孔加入100μL TMB显色液，室温避光孵育3-10分钟；然后每孔加入50μL终止液(2M HCl)，通过多功能酶标仪(Varioskan，Thermo)读取OD450值。利用GraphPad Prism 9软件对原始数据进行分析，结果如图1所示，mAb2117、mAb2126、mAb2128、mAb2133、mAb2138、mAb2164、mAb2170与HER2结合活性较强且与人源ErbB/HER家族其它成员无交叉结合反应。
1.5抗HER2嵌合抗体与表达HER2的肿瘤细胞的结合活性检测
利用流式细胞术检测抗HER2嵌合抗体与NCI-N87细胞的结合活性。具体方法如下：收集NCI-N87细胞，用预冷的FACS缓冲液(PBS+1％ BSA)清洗一次后将细胞重悬至6～10×106细胞/mL，并以50μL/孔加入至96孔U型板中，随后加入50μL/孔梯度稀释的待测嵌合抗体或阳性对照抗体Trastuzumab(以FACS缓冲液为阴性空白对照)，于冰上静置孵育60分钟。加入预冷的FACS缓冲液清洗细胞两次后以每孔100μL加入1:1,000稀释于FACS缓冲液中的抗-人IgG(H+L)-AF488抗体(Jackson Immuno)，于冰上避光孵育40分钟。加入预冷的FACS缓冲液清洗细胞两次后每孔加入100μL预冷的FACS缓冲液重悬细 胞，随后通过流式细胞仪(NovoCyte 3005，Agilent)检测。结果如图2所示，mAb2117、mAb2126、mAb2164、mAb2170抗体与NCI-N87细胞的结合活性较强。
1.6抗HER2嵌合抗体的抗原结合表位分析
根据人源与鼠源HER2胞外区亚结构域的结构，将人、鼠HER2不同亚结构域的基因序列进行重组拼接，同源重组连接至pcDNA3.1载体以构建人-鼠嵌合的HER2胞外区重组蛋白表达质粒(图3A)，瞬转表达获得人-鼠嵌合的HER2胞外区重组蛋白上清(各重组蛋白的C端均带有6×His标签)。以所述人-鼠嵌合重组蛋白为抗原，通过ELISA法检测嵌合抗体在HER2胞外区内的结合表位。具体方法如下：将待测嵌合抗体分别用PBS稀释后，以100μL/孔加入到96孔ELISA板中，4℃孵育过夜。PBS洗板后每孔加入200μL封闭液(含有1％ BSA的PBST)，室温静置孵育1小时。PBST洗板后每孔加入100μL以1:200稀释于封闭液中的人HER2、小鼠HER2、或人-鼠嵌合HER2重组蛋白，包括HER2(D1)，HER2(D1-2)，HER2(D1-3)，HER2(D2)，HER2(D3)、HER2(D4)，其中括号内的后缀表示HER2嵌合抗原中的一个或数个胞外区亚结构域的人源序列被相应的鼠源序列所置换，例如D1表示胞外区亚结构域1被鼠源序列所置换。室温静置孵育2小时后，PBST洗板并每孔加入100μL以1:10,000稀释于封闭液中的抗-6×His-HRP抗体(Proteintech)，室温静置孵育1小时。PBST洗板后每孔加入100μL TMB显色液，室温避光孵育3-10分钟后每孔加入50μL终止液(2M HCl)，然后利用多功能酶标仪(Varioskan，Thermo)读取OD450值。利用GraphPad Prism 9软件对原始数据进行分析。检测结果如图3B所示，mAb2117的识别表位位于HER2胞外区的D3，mAb2126、mAb2164和mAb2170的识别表位位于HER2胞外区的D1，mAb2128的识别表位位于HER2胞外区的D4。
1.7抗HER2嵌合抗体的抗原结合竞争性分析
为了进一步验证各嵌合抗体与HER2结合的表位是否有重叠或交叉，通过竞争ELISA法检测各嵌合抗体间的抗原结合竞争活性。具体方法如下：用PBS稀释HER2胞外区重组蛋白，并以100μL/孔加入到96孔ELISA板中，4℃孵育过夜。PBS洗板后每孔加入200μL封闭液(含有1％ BSA的PBST)，室温静置孵育1小时。PBST洗板后以50μL/孔加入梯度稀释于封闭液中的各待测嵌合抗体或对照抗体Trastuzumab(起始浓度为50μg/mL，3倍梯度稀释，共7个浓度梯度)，室温静置孵育1小时后每孔加入50μL经生物素标记的各嵌合抗体(终浓度均为0.5μg/mL)，继续室温静置孵育1小时。PBST洗板后以每孔100μL加入1:10,000稀释于封闭液中的HRP-Streptavidin(Jackson Immuno)，室温孵育1小时。PBST洗板后每孔加入100μL TMB显色液，室温避光孵育3-10分钟后每孔加入50μL终止液(2M HCl)，然后利用多功能酶标仪(Varioskan，Thermo)读取OD450值。检测结果显示，mAb2164与mAb2170完全竞争结合HER2胞外区重组蛋白(图4A)，mAb2164与mAb2117、mAb2126部分竞争结合HER2胞外区重组蛋白，mAb2164与Trastuzumab不竞争结合HER2胞外区重组蛋白(图4A)；mAb2164与mAb2128不竞争结合HER2胞外区重组蛋白，mAb2117与mAb2126、mAb2128不竞争结合HER2胞外区重组蛋白(图4B)。1.8抗HER2嵌合抗体的抗原结合动力学分析
利用分子相互作用仪(ForteBio,型号R8)检测各嵌合抗体与HER2胞外区重组蛋白的结合动力学。具体方法如下：用PBS将待测嵌合抗体mAb2117、mAb2126、mAb2128、mAb2164、mAb2170稀释至5μg/mL，并分别以200μL每孔加入96孔黑壁板内；然后用PBS将HER2胞外区重组蛋白稀释至200nM，并作1:2梯度稀释后以200μL每孔加入96孔黑壁板内。将含有待测样品的96孔黑壁板和Protein A检测探针放置于检测仪中，设置程序使Protein A探针捕获抗体，随后进行与抗原的结合与解离检测；每一次结合与解离循环完毕后将探针浸没于10mM甘氨酸缓冲液(pH 1.5)中使探针再生，然后再开始下一循环检测。待检测完毕后使用分析软件对相应抗体与抗原之间的结合常数和解离常数进行拟合计算，并计算得到亲和力常数(KD)数值。结果如表4所示，嵌合抗体mAb2117、mAb2126、mAb2128、mAb2164、mAb2170与HER2胞外区重组蛋白的结合亲和力常数(KD)值均约为10-9M。
表4.嵌合抗体与HER2胞外区重组蛋白的结合动力学分析结果

Full R2表示拟合曲线与实测曲线的相似度。
1.9抗HER2嵌合抗体对HER2中Y1248位点磷酸化的作用
已有发表的研究结果显示Trastuzumab能够诱导心肌细胞中HER2胞内结构域的Y1248位点发生磷酸 化并由此引发一系列的信号转导级联反应，最终导致心肌细胞的稳态遭到破坏(Mohan等，Molecular Cancer Therapeutics 2016,15:1321-1331)，这一机制可能是Trastuzumab导致心脏毒性的原因之一，因此本实施例通过检测抗HER2抗体能否诱导SKBR-3细胞的HER2-Y1248位点磷酸化来评估其是否具有类似Trastuzumab的心脏毒性。具体方法如下：收集SKBR-3细胞并重悬于RPMI-1640无血清培养基(含0.1％BSA)中，然后接种至6孔板，37℃培养过夜。于各孔中分别加入终浓度为4μg/mL的待测抗体或对照抗体(Trastuzumab、Pertuzumab)，37℃孵育30分钟。收集各孔细胞于1.5-mL EP管中，并用预冷的PBS清洗2次后加入150μL RIPA细胞裂解液(Porteintech)进行全蛋白提取。4℃高速离心10分钟收集裂解液上清，通过BCA法检测各裂解液上清中蛋白浓度，并加入还原缓冲液进行蛋白电泳样品制备，随后进行SDS-PAGE凝胶电泳。电泳结束后以80V转膜90分钟将蛋白条带转印至PVDF膜上，随后在室温条件下用5％脱脂牛奶对PVDF膜进行封闭。以1:1000的稀释比例加入兔抗人HER2(Y1248)抗体(CST)或兔抗人HER2抗体(Abcam)，于4℃孵育过夜。用PBST清洗PVDF膜3次后加入1:5000稀释于5％脱脂牛奶中的山羊抗兔IgG-HRP抗体(Jacskon Immuno)，室温下孵育1小时。用PBST清洗PVDF膜3次后加入显影液并利用成像仪(Bio-Rad)进行检测。结果如图5所示，与阳性对照Trastuzumab相比，嵌合抗体mAb2117和mAb2126没有诱导HER2-Y1248的磷酸化，说明它们可能不具有类似Trastuzumab的心脏毒性。
1.10抗HER2嵌合抗体对NRG-1诱导的HER2下游信号转导通路激活的影响
NRG-1能够诱导HER2的异源二聚化并激活下游信号转导通路，其中AKT磷酸化是其下游信号转导级联反应中的一个标志性事件，本实施例通过检测抗体对NRG-1诱导的AKT磷酸化的抑制作用来评估它们对HER2受体信号转导通路的影响。具体方法如下：将T47D细胞接种至6孔板中，待细胞生长至约80％融合状态后，弃去培养基并加入不含血清的RPMI-1640饥饿培养过夜。于各孔中分别加入终浓度为100nM的待测抗体或对照抗体(Trastuzumab、Pertuzumab)，37℃孵育20分钟后，于各孔中加入终浓度为100ng/mL的NRG-1(NOVUS)并继续孵育10分钟。用预冷的PBS清洗细胞2次后，按照本实施例第1.9项所述方法进行全蛋白提取以及Western-blot检测，所用一抗为兔抗p-AKT(Ser465)抗体(CST)或兔抗pan-AKT抗体(CST)，二抗为山羊抗兔IgG-HRP抗体(Jacskon Immuno)。结果如图6所示，与阳性对照抗体Pertuzumab相比，嵌合抗体mAb2117和mAb2126对NRG-1诱导的AKT磷酸化无抑制作用，说明它们不影响NRG-1诱导的HER2异源二聚化及其下游信号转导通路的激活。
实施例2抗HER2嵌合抗体mAb2117的人源化及优化改造
2.1抗HER2嵌合抗体mAb2117的人源化
经MOE软件分析，采用CDR移植的方法将嵌合抗体mAb2117的重链骨架区序列用Germline IGHV3-23的骨架区序列替换，标记为Hu2117-H1；mAb2117的轻链骨架区序列用Germline IGKV1-39的骨架区序列替换，标记为Hu2117-K1。将mAb2117嵌合抗体及其人源化抗体分别构建成为hIgG1Fc的单链抗体融合蛋白(scFv-hIgG1Fc融合蛋白)，通过基因序列合成方式获得scFv抗体基因片段，分别为人源化单链抗体Hu2117HK和Hu2117KH以及嵌合单链抗体对照Mu2117HK和Mu2117KH，其中HK代表单链抗体的结构为“(N端)重链可变区核酸序列-连接子(G4S)3-轻链可变区核酸序列(C端)”，KH代表单链抗体的结构为“(N端)轻链可变区核酸序列-连接子(G4S)3-重链可变区核酸序列(C端)”。将scFv基因片段分别同源重组于pcDNA3.1(含人源IgG1Fc序列)载体中，构建抗体表达质粒，提取表达质粒后分别瞬转到ExpiCHO-S细胞(Thermo)中进行无血清培养，利用AKTA Pure系统进行Protein A亲和层析纯化培养上清中的抗体，获得单链抗体融合蛋白。利用ForteBio进行单链抗体融合蛋白与HER2重组蛋白的结合动力学分析，检测结果如表5所示，所述人源化抗体基本保留了与HER2的结合活性，亲和力常数(KD)约为10-7M。
表5.单链抗体融合蛋白与HER2重组蛋白的结合动力学分析结果

Full R2表示拟合曲线与实测曲线的相似度。
根据实施例1中第1.5项所述方法，利用流式细胞术检测单链抗体融合蛋白Hu2117HK和Hu2117KH与BT474细胞的结合活性，其中嵌合抗体mAb2117和Trastuzumab为阳性对照，结果显示mAb2117、Hu2117HK、Hu2117KH均能与BT474细胞结合，其中Hu2117HK结合BT474细胞的活性略高于Hu2117KH(数据未显示)。
2.2抗HER2人源化抗体Hu2117HK的单点或多点突变优化
利用在线软件abYsis(http://abysis.org/abysis/index.html)和MOE软件，对人源化抗体Hu2117HK的轻、重链可变区序列中人源化程度较低的氨基酸残基、潜在的稳定性风险位点、以及骨架区中的鼠源氨基酸残基进行分析后，共设计了50个单点/多点突变拟提高抗体的人源化程度、抗原结合亲和力和/或稳定性。具体地，在单链抗体Hu2117HK序列中通过PCR分别引入所设计的50个单点/多点突变，并进行表达获得单链抗体突变体蛋白。
利用ForteBio分子相互作用仪检测上述50个单链抗体突变体及其母本单链抗体Hu2117HK的瞬转表达量。具体方法如下：于ExpiCHO-S细胞瞬转表达后收获培养上清，以200μL/孔加入到96孔黑板中，将待测样品及Protein A探针置于ForteBio作用仪内，利用预设的定量程序检测培养上清中各抗体分子与Protein A探针的结合速率；每一循环完毕后将探针浸没于10mM甘氨酸缓冲液(pH 1.5)中使探针再生，然后开始下一循环检测。检测完毕后使用分析软件根据标准曲线进行计算得到各单链抗体的表达量。同时，按照实施例1中第1.8项所述方法，利用ForteBio分析各单链抗体突变体与HER2的结合动力学。
各单点/多点突变位点与突变氨基酸、瞬转表达量以及抗原结合动力学分析结果如表6所示。
表6.单链抗体Hu2117HK的单点/多点突变、瞬转表达量、以及与HER2的结合动力学分析结果


L为轻链；H为重链；N/A表示不适用；Full R2表示拟合曲线与实测曲线的相似度。
按照实施例1中第1.5项所述方法，利用流式细胞术检测含有单点/多点突变的单链抗体与BT474细胞以及RT-112细胞的结合活性，其中阳性对照抗体为母本单链抗体Hu2117HK。结果如图7所示，与母本单链抗体Hu2117HK相比，大多数单链抗体突变体都保留了对BT474细胞的结合活性，某些抗体的细胞结合活性甚至略有提高，这些抗体包括Hu2117HK-Mu01、-Mu02、-Mu03、-Mu04、-Mu05、-Mu06、-Mu08、-Mu09、-Mu10、-Mu11、-Mu12、-Mu13、-Mu17、-Mu20、-Mu21、-Mu23、-Mu24、-Mu25、-Mu27、-Mu28、-Mu32、-Mu33、-Mu36、-Mu37、-Mu38、-Mu39、-Mu40、-Mu45、-Mu46、-Mu47、-Mu48、-Mu49、-Mu50(图7A)。同时，与RT-112细胞的结合结果如图7B所示，与母本单链抗体Hu2117HK相比，大多数单链抗体突变体都保留了对RT-112细胞的结合活性，这些抗体包括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、-Mu50。
2.3抗HER2人源化抗体Hu2117HK的组合突变优化
将从上述优选的单点/多点突变进行组合并引入到人源化单链抗体Hu2117HK的可变区序列中，构建含有不同组合突变的抗体分子Hu2117-HK201～-HK203(简称HK201～HK203)，以及Hu2117-HK301～-HK312(简称HK301～HK312)。按照本实施例中第2.2项所述方法进行单链抗体样品的制备，并采用ForteBio检测各突变体以及母本单链抗体Hu2117HK的瞬转表达量。各抗体分子包含的组合突变及瞬转表达量如表7所示。
表7.单链抗体Hu2117HK的组合突变优化以及瞬转表达量

按照实施例1中第1.5项所述方法，利用流式细胞术检测含有组合突变的单链抗体及其母本单链抗体Hu2117HK分别与BT474细胞和RT-112细胞的结合活性。结果显示，在10μg/mL的饱和浓度下，HK201、HK202、HK203抗体与BT474细胞和RT-112细胞均能结合，且结合活性与母本抗体Hu2117HK相当；HK301、HK303、HK304、HK305、HK306、HK309、HK310均能与RT-112细胞结合，且与母本抗体Hu2117HK的结合活性相当(数据未显示)。
为了进一步鉴定经组合突变优化后的抗体的细胞结合活性，优选HK203、HK303、HK304、HK309、和HK310抗体，按照实施例1中第1.5项所述方法，将抗体梯度稀释后利用流式细胞术检测其与BT474细胞以及RT-112细胞的结合，其中对照抗体为母本单链抗体Hu2117HK。结果如表8及图8所示，这些优化的人源化抗体均能与BT474细胞以及RT-112细胞结合，且结合活性与母本抗体Hu2117HK相当。
表8.单链抗体Hu2117HK的组合突变优化分子与BT474细胞以及RT-112细胞的结合活性
实施例3抗HER2嵌合抗体mAb2126的人源化及优化改造
3.1抗HER2嵌合抗体mAb2126的人源化
经MOE软件分析，采用CDR移植的方法将嵌合抗体mAb2126的重链骨架区序列用Germline IGHV4-4或IGHV7-4的骨架区序列替换，分表标记为Hu2126-H1、Hu2126-H2；mAb2126的轻链骨架区序列用Germline IGKV1-39的骨架区序列替换，标记为Hu2126-K1。通过基因序列合成方式获得人源化抗体可变区基因片段，将轻、重链基因片段分别与线性化的表达载体pcDNA3.1连接以构建抗体表达质粒(重链可变区[VH]与含人源IgG1恒定区的pcDNA3.1载体连接，轻链可变区[VL]与含人源CK恒定区的pcDNA3.1载体连接)，按照常规方法提取表达质粒后分别瞬转到ExpiCHO-S细胞(Thermo)中进行无血清表达培养，培养上清利用AKTA Pure系统进行Protein A亲和层析纯化，获得人源化抗体Hu2126-H1K1和Hu2126-H2K1。
按照实施例1中第1.8项所述方法，利用ForteBio检测各人源化抗体与HER2的结合动力学。结果如表9所示，经CDR移植而获得的人源化抗体保留了与HER2的结合活性，其中Hu2126-H2K1的结合亲和力与母本嵌合抗体mAb2126的相当。
表9.嵌合抗体mAb2126人源化(CDR移植)前后的结合动力学分析结果
Full R2表示拟合曲线与实测曲线的相似度。
3.2抗HER2人源化抗体Hu2126-H2K1的单点突变优化
利用在线软件abYsis(http://abysis.org/abysis/index.html)和MOE软件对人源化抗体Hu2126-H2K1的 轻、重链可变区序列中人源化程度相对较低的氨基酸残基、潜在的分子稳定性风险位点、以及骨架区中的鼠源氨基酸残基进行分析后，共设计了63个单点突变拟提高抗体的人源化程度、抗原结合亲和力，和/或稳定性。具体地，在人源化抗体Hu2126-H2K1序列中通过PCR分别引入所设计的63个单点突变，并构建表达质粒，然后将抗体轻、重链表达质粒组合后瞬转ExpiCHO-S细胞进行抗体表达，获得63个含单点突变的抗体突变体表达上清。按照实施例2第2.2项所述方法，利用ForteBio检测各抗体突变体在瞬转培养3天后的表达量以及与HER2的结合动力学。
结果如表10所示，与母本抗体Hu2126-H2K1相比，所设计的63个单点突变中共有42个不显著影响抗体的抗原结合亲和力，包括抗体轻链中的17个单点突变：2126-Hu-L02、-L05、-L06、-L07、-L08、-L09、-L10、-L11、-L12、-L13、-L14、-L15、-L16、-L17、-L18、-L19、-L21，以及抗体重链中的25个单点突变：2126-Hu-H22、-H23、-H24、-H25、-H27、-H28、-H29、-H31、-H32、-H33、-H34、-H35、-H36、-H37、-H38、-H39、-H40、-H42、-H43、-H44、-H45、-H46、-H50、-H54、-H59。
表10.Hu2126-H2K1人源化抗体的单点突变、瞬转表达量以及与HER2的结合动力学分析结果


L为轻链；H为重链；N/A代表不适用；Full R2表示拟合曲线与实测曲线的相似度。
3.3抗HER2人源化抗体Hu2126-H2K1的组合突变优化
优选上述轻链中的单点突变进行组合后引入到Hu2126-K1中，以及优选上述重链中的单点突变进行组合后引入到Hu2126-H2中，分别构建轻链突变组合表达质粒和重链突变组合表达质粒，然后将轻、重链表达质粒进行配对并瞬转ExpiCHO-S细胞，培养表达和纯化后获得Hu2126-H2K1-L71-H72b-Mu10～-Mu19(简称7172b-Mu10～-Mu19)，共计10个抗体突变体。按照实施例1中第1.8项所述方法，利用ForteBio检测含组合突变的人源化抗体与HER2的结合动力学。
各抗体突变体所含的组合突变以及抗原结合动力学分析结果如表11所示，抗体7172b-Mu10、7172b-Mu14、7172b-Mu17和7172b-Mu19与HER2的结合亲和力明显高于母本抗体Hu2126-H2K1。
表11.抗体Hu2126-H2K1的组合突变优化，以及与HER2的结合动力学分析结果


Full R2表示拟合曲线与实测曲线的相似度。
实施例4抗HER2互补性双特异抗体的构建、优化及功能活性鉴定
4.1抗HER2互补性双特异抗体的构建
本实施例选择能够分别识别HER2胞外区不同表位且彼此之间互不竞争的一对单特异性抗体(Hu2117-HK304和Hu2126-H2K1-L71-H72b-Mu14)进行示例性互补性双特异抗体的构建。如图9所示，双抗构型为DVD-IgG结构，Fv结构域为Hu2117-HK304的可变区序列，IgG结构域为Hu2126-H2K1-L71-H72b-Mu14序列，其中Fv结构域的重链可变区与IgG结构域的重链通过连接子(G4S)1相连，Fv结构域的轻链可变区与IgG结构域的轻链通过连接子(G4S)3相连，同时为了避免抗体Fc区介导的效应子功能可能对非靶细胞造成杀伤作用，通过在双抗分子Fc区中引入L234F/L235E/P331S(EU Numbering)突变(简称“TM突变”)以降低其与FcγRs和C1q的结合，从而消除Fc效应子功能，所构建的互补性双特异抗体命名为04BS-109-WT。将04BS-109-WT双抗表达质粒瞬转ExpiCHO-S细胞进行抗体蛋白表达，并通过Protein A亲和层析纯化，纯化过程中发现04BS-109-WT双抗分子在洗脱时(洗脱液不含NaCl)发生胶体状沉淀，洗脱产物在溶液中呈浑浊状，但在加入0.1M NaCl后胶状沉淀逐渐溶解，抗体溶液最终呈澄清状。
4.2抗HER2互补性双特异抗体的优化
通过MOE软件分析，在互补性双特异抗体04BS-109-WT的Fv结构域的HCDR2序列中设计了一系列突变拟改善双抗分子的稳定性，所设计的突变如表12所示并分别命名为04BS-1123-ST01～-ST06。通过PCR在04BS-109-WT分子序列中引入突变，将构建的各双抗突变体表达质粒瞬转ExpiCHO-S细胞并进行无血清培养以表达抗体蛋白。按照实施例2第2.2项所述方法，利用ForteBio检测培养上清中的抗体表达量，结果显示04BS-1123-ST03抗体的表达量过低(＜30mg/L)，其余双抗突变体在瞬转培养10天后的表达量均在300mg/L以上。在经过Protein A亲和层析纯化后，SEC-HPLC分析显示各双抗突变体的单体纯度均接近100％。然而，纯化过程中发现各双抗突变体的分子稳定性差异明显(如表12所示)，其中04BS-1123-ST06双抗在Protein A亲和层析纯化洗脱过程中，无论洗脱液是否含有0.1M NaCl，样品溶液都呈澄清状，表明04BS-1123-ST06双抗的分子稳定性似优于其它突变体以及母本抗体04BS-109-WT。
表12.示例性抗HER2互补性双特异抗体04BS-109-WT及其突变体的分子稳定性表现

注：下划线氨基酸残基表示在Hu2117-HK304的HCDR2序列中引入的氨基酸突变。
4.3抗HER2人源化抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14的细胞竞争结合检测
由04BS-1123-ST06的Fv结构域序列衍生而来的抗HER2单特异性抗体被命名为Hu2117-HK304-06。为了进一步确认优化后的抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14是否保留了其母本抗体(嵌合抗体mAb2117与mAb2126)不竞争结合HER2的特性，本实验按照实施例1中第1.5项所述方法， 将梯度稀释自800μg/mL的这两个优化抗体分别与8μg/mL生物素标记的Hu2117-HK304-06抗体等体积混合后与BT474细胞进行孵育，然后以AF488标记的链酶亲和素(Jackson Immuno)为二抗，通过流式细胞术检测这两个抗体在表达HER2的肿瘤细胞上的竞争抑制活性。结果如图10所示，Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14之间不竞争结合表达于细胞表面的HER2。
4.4抗HER2互补性双特异抗体的细胞内吞反应以及溶酶体转运/降解效率分析
本实施例利用流式细胞术对上述示例性抗HER2互补性双特异抗体在表达不同水平HER2的细胞(包括BT474、JIMT-1、RT-112)中的内吞率进行了测定。具体方法如下：细胞经胰蛋白酶消化收集后，用预冷的FACS缓冲液清洗并重悬至6～10×106细胞/mL；将细胞悬液以300μL/孔转移至96孔U型深孔板中，随后在各孔中加入300μL待测抗体(抗体终浓度为20μg/mL)，混匀后在冰上孵育30～60分钟；用预冷的FACS缓冲液清洗两次后，各孔细胞以600μL/孔预冷的FACS缓冲液充分重悬，随后以100μL/孔将细胞悬液均匀分装至5块96孔U型板中，任取其中一块板作为对照组置于冰上孵育，其余4块板作为实验组置于37℃孵育并分别于30分钟、1小时、2小时和4小时后将其中一块板转移至冰上冷却以终止内吞反应；待全部孔板孵育完毕后，各孔中加入预冷的FACS缓冲液清洗细胞2次，并加入100μL/孔固定液(2％多聚甲醛)，置于4℃过夜；在各板中加入预冷的FACS缓冲液清洗2次后以100μL/孔加入1:1000稀释于预冷的FACS缓冲液的AF488标记的抗-人IgG(H+L)抗体(Jackson Immuno)，重悬细胞后在冰上避光孵育40分钟；加入预冷的FACS缓冲液清洗细胞2次，以100μL/孔FACS缓冲液重悬细胞后利用流式细胞仪进行检测。抗体的内吞率(百分比值)以如下公式进行计算：(MFI对照组-MFI实验组)/MFI对照组×100％。结果如图11所示，相较于对照抗体Trastuzumab以及相关的单特异性抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14，抗HER2互补性双特异抗体04BS-1123-ST04、04BS-1123-ST05、04BS-1123-ST06及其母本抗体04BS-109-WT在BT474、JIMT-1和RT-112细胞中均能诱发更加快速高效的内吞，其中，所述互补性双特异抗体在JIMT-1和RT-112细胞中内吞率可达60％左右，在BT474细胞中内吞率可高达80％左右。
为了进一步验证抗HER2互补性双特异抗体在内吞后被转运入溶酶体中，本实施例利用共聚焦显微镜方法对抗体的亚细胞定位进行了检测。具体方法如下：收集SKBR-3细胞，铺入5块8孔腔室载玻片(Thermo)中进行培养；待细胞生长至均一单层后，弃去培养基并用PBS润洗一次；在腔室载玻片的小室中分别加入500μL浓度为10μg/mL的抗HER2互补性双特异抗体04BS-1123-ST06、对照抗体Trastuzumab、或人IgG同型对照抗体(如WO2003106496A1中所述的抗HIV/gp120抗体)，于冰上孵育1小时；任选其中一块腔室载玻片作为0小时对照，用预冷的PBS润洗一次后加入2％多聚甲醛进行固定，其余4块则置于37℃分别孵育0.5小时、2小时、4小时和8小时后用预冷PBS润洗一次，并加入2％多聚甲醛于4℃静置固定过夜；用PBST(PBS含0.05％ Tween-20)润洗两次后，于各小室中加入250μL固定破膜液(PBS含2％山羊血清和0.5％ Triton X-100)室温孵育10分钟；用PBST润洗两次，在各小室中加入250μL稀释于FACS缓冲液(PBS含2％ FBS)的小鼠抗-LAMP1抗体(1:50稀释，BD)和AF488标记的山羊抗人IgG抗体(2μg，Molecular Probes)，于室温避光孵育1小时；用PBST润洗两次，于各小室中加入250μL含AF647标记的山羊抗小鼠IgG抗体(1:250稀释，Molecular Probes)的FACS缓冲液，于室温避光孵育1小时；用PBST润洗两次，于各小室加入终浓度为1μg/mL的DAPI染色1分钟；用PBST润洗1次后，滴加封板液(Thermo)进行封板，并于室温避光静置过夜；利用共聚焦显微镜进行扫描分析。结果如图12所示，人IgG同型对照抗体孵育的细胞在各时间点都未检测到绿色荧光信号，表明细胞没有非特异性的抗体结合；阳性对照抗体Trastuzumab孵育的细胞能检测到绿色荧光信号，并且在各时间点没有明显差别，均呈环状均匀分布于细胞膜上，与红色荧光所标记的溶酶体未出现重合，说明Trastuzumab能够结合SKBR-3细胞上的HER2，但未发生显著的内吞和溶酶体转运；而抗HER2互补性双特异抗体04BS-1123-ST06孵育的细胞随着处理时间的进展，绿色荧光信号逐渐由细胞膜上转向细胞内，并与红色荧光标记的溶酶体出现重合(呈黄色，如图中箭头所示)，之后黄色荧光信号强度随着时间的推移逐渐减弱甚至完全消失，说明内吞的抗HER2互补性双特异抗体04BS-1123-ST06被转运进了溶酶体内并发生降解，其溶酶体转运效率几乎为100％。
通过Western-blot方法检测HER2的降解，更进一步验证了抗HER2互补性双特异抗体在结合细胞表面HER2后被内吞以及转运进溶酶体内被降解的结局。具体方法如下：收集BT474细胞，用完全培养基(含10％ FBS的RPMI-1640培养基)重悬至1×106细胞/mL，以500μL/管分装细胞悬液至1.5-mL EP管中；用完全培养基将待测抗体(包括互补性双特异抗体04BS-1123-ST06，对照抗体Trastuzumab，以及所述互补性双特异性抗体对应的单特异性抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14)稀释至300μg/mL，随后以100μL/管分别加入上述EP管内(抗体终浓度为50μg/mL)；将各EP管置于37℃/5％ CO2孵箱中静置孵育24小时，然后离心收集细胞并用预冷的PBS清洗2次；以100μL/管加入RIPA裂解液(Proteintech)重悬细胞并于冰上孵育30分钟；各管于4℃高速离心10分钟后收集裂解液上清，利用BCA试剂盒(Thermo)对提取液中的蛋白浓度进行定量；根据蛋白浓度，用RIPA裂解液将各提取液中蛋白浓 度稀释至0.125μg/μL，随后加入含DTT的5×SDS-PAGE样品缓冲液，并于95℃加热5分钟以制备SDS-PAGE上样样品。将处理好的样品以10μL/道(即1μg/lane)加样至10％ SDS-PAGE凝胶上进行电泳，随后以80V的电压将蛋白转印至PVDF膜上；用5％脱脂牛奶室温封闭PVDF膜1小时，随后在55kDa分子量Marker处将膜裁剪成两半，其中上半部分加入1:5000稀释的兔抗人HER2抗体(CST)并于4℃孵育过夜，下半部分加入1:10,000稀释的鼠抗人GAPDH抗体(Proteintech)并于室温孵育1小时；孵育完成后，用PBST清洗3次，随后各自加入1:5000稀释的山羊抗兔IgG-HRP抗体(Jackson Immuno)和山羊抗小鼠IgG-HRP抗体(Jackson Immuno)，室温孵育1小时；用PBST清洗3次，随后利用ECL显影液(南京诺唯赞)进行曝光显影。结果如图13所示，互补性双特异抗体04BS-1123-ST06能够有效诱导降解BT474细胞的HER2，而对照抗体则不能诱发HER2的降解。
本实施例还检测了互补性双特异抗体04BS-1123-ST06对BT474细胞增殖的影响。具体方法如下：在96孔白壁板中接种BT474细胞(10,000细胞/孔)，并加入梯度稀释的待测抗体(包括04BS-1123-ST06、Trastuzumab、Pertuzumab、Hu2117-HK304-06、Hu2126-H2K1-L71-H72b-Mu14)，细胞于37℃/5％ CO2孵育5天后加入检测试剂CellCounting-Lite 2.0Luminescent Reagent(南京诺唯赞)，并用多功能酶标仪(Varioskan,Thermo)读取化学发光值，利用GraphPad Prism 9软件对实验数据进行分析。结果如图14所示，互补性双特异抗体04BS-1123-ST06能够有效抑制BT474细胞的增殖，但是对应的单特异性抗体均没有抑制BT474细胞增殖的活性。由于BT474细胞的增殖依赖于其过表达的HER2在细胞膜上形成的同源二聚体所激活的信号转导机制，因此推断所述互补性双特异抗体的细胞增殖抑制活性是因细胞表面HER2被内吞/降解而使HER2同源二聚体无法形成所致。
4.5抗HER2互补性双特异抗体对NRG-1诱导的HER2/HER4二聚化的影响
利用Promega的NanoBiT结构互补报告分子系统对抗HER2互补性双特异抗体是否会影响NRG-1诱导的HER2/HER4二聚化展开了进一步验证。NanoBiT系统由一个LgBiT亚基和一个SmBiT亚基组成，可分别与待测蛋白融合；若两个待测蛋白相互作用形成二聚体，LgBiT则会和SmBiT结构互补形成功能性荧光素酶，进而与底物反应产生发光信号。在本实施例中，首先通过分子克隆技术将编码HER2和HER4膜外区及跨膜区的DNA序列插入NanoBiT系统pBiT1.3-C和pBiT2.3-C载体中，然后将这两个质粒共转染U2OS细胞；于转染24小时后收集细胞，并铺于96孔白壁板中，37℃培养过夜。将互补性双特异抗体04BS-1123-ST06、人源化抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14、嵌合抗体mAb2117和mAb2126、以及对照抗体Trastuzumab和Pertuzumab进行梯度稀释，随后按照1:1的体积比与NRG-1混匀待用(NRG-1的终浓度为1nM)。于96孔白壁板中加入Nano-Glo Live Cell Reagent(Promega)，并读取此时的化学发光值作为本底，随后在各孔中加入上述制备好的样品，37℃孵育6分钟后读取化学发光值，并利用GraphPad Prism 9软件对实验数据进行分析。结果如图15所示，对照抗体Trastuzumab和Pertuzumab能够显著抑制NRG-1诱导的HER2/HER4二聚化，而嵌合抗体mAb2117和mAb2126、单特异性人源化抗体Hu2117-HK304-06和Hu2126-H2K1-L71-H72b-Mu14(图15A)以及基于这两个单特异性人源化抗体所构建的互补性双特异抗体04BS-1123-ST06(图15B)均不具有抑制作用，该结果进一步说明所述抗HER2互补性双特异抗体不会影响HER2的正常生物学功能，因而其在体内诱发心脏毒性的风险极低。4.6抗HER2互补性双特异抗体对NRG-1诱导的HER2下游信号转导通路激活的影响
人体细胞诱导的多能干细胞(Induced Pluripotent Stem Cell,简称iPSC)以及相关培养试剂盒购自Fujiflim Cellular Dynamics。按照生产商提供的指导说明书所述的方法，将iPSC铺入6孔板并置于37℃、5％CO2条件下培养一周时间使细胞完全分化为人心肌细胞(iCell Cardiomyocytes)。按照实施例1中第1.10项所述方法检测抗HER2互补性双特异抗体04BS-1123-ST06对NRG-1诱导的人心肌细胞内AKT磷酸化的影响。结果如图16所示，不同于阳性对照抗体Pertuzumab，04BS-1123-ST06对人心肌细胞中NRG-1诱导的AKT磷酸化无抑制作用，说明所述互补性双特异抗体不影响心肌细胞中NRG-1诱导的HER2二聚化及其下游信号转导通路的调控。
4.7抗HER2互补性双特异抗体的Fc效应子功能改造
为了降低因Fc效应子功能而带来的潜在安全性风险，本实施例在所述抗HER2互补性双特异抗体04BS-1123-ST06的Fc区中引入了三个点突变：L234F/L235E/P331S(简称TM突变)，以降低抗体与Fc受体及C1q的结合活性。通过ForteBio检测证实了含有TM突变的互补性双特异抗体04BS-1123-ST06与FcγRI、FcγRIIa(167H)、FcγRIIb、FcγRIIIa(176V)、FcγRIIIa(176F)及C1q均无明显结合反应，然而，所述互补性双特异抗体与FcRn的结合亲和力相较于Trastuzumab则无显著变化(数据未显示)。
此外，本实施例还以表达FcγRIIIa-176V受体和NFAT反应元件调控的萤火虫荧光素酶报告基因的Jurkat细胞(Promega)为效应细胞，以过表达HER2的BT474细胞为靶细胞，并以Trastuzumab为阳性对照抗体，进一步对含有TM突变的互补性双特异抗体04BS-1123-ST06是否具有ADCC活性进行了验证。具体方法如下：离心收集BT474细胞，用ADCC检测缓冲液(含有0.5％ FBS的RPMI-1640培养基)重悬后，以1.5×104细胞/孔加入96孔白壁板中。用ADCC检测缓冲液梯度稀释待测抗体，以25μL/孔加入 上述96孔板中。离心收集效应细胞并用ADCC检测缓冲液重悬后，以1.5×105细胞/孔加入上述96孔板中，然后于37℃、5％ CO2下孵育过夜。第二天将96孔板置于室温平衡，然后加入检测试剂Bio-Lite Luciferase Reagent(南京诺唯赞)，利用多功能酶标仪(Varioskan,Thermo)读取化学发光值，并利用GraphPad Prism 9软件对实验数据进行分析。检测结果如图17所示，不同于阳性对照抗体Trastuzumab，04BS-1123-ST06即使在最高浓度下也未诱导任何荧光素酶的表达，表明在Fc区引入TM突变的抗HER2互补性双特异抗体不具有ADCC活性。
实施例5抗HER2互补性双特异抗体ADC的制备与功能活性鉴定
5.1抗HER2互补性双特异抗体-药物偶联物的制备
本实施例将抗HER2互补性双特异抗体04BS-1123-ST06通过可裂解接头(例如，Mal-(PEG)2-VC-PAB、Mal-(PEG)2-GGFG)与具有抗肿瘤活性的小分子毒素化合物艾日布林(Eribulin)偶联以制备抗HER2互补性双特异ADC。所述艾日布林是根据ZL201910197071.8或ZL201910509222.9，以及WO1999065894中所述方法制备得到。
药物有效载荷(Payload)Mal-(PEG)2-VC-PAB-Eribulin的制备参考WO2017151979实施例2.1.1中所述的方法。
药物有效载荷Mal-(PEG)2-GGFG-Eribulin的制备方法如下：将NH2-(PEG)2-COOH和马来酸酐加入醋酸中，加热回流过夜，旋蒸除去醋酸，残留物经HPLC制备、冻干即得Mal-(PEG)2-COOH。以2Cl Trt Resin为固相载体，用20％哌啶/DMF(v/v)溶液脱除Fmoc保护，再以HOBT/DIC为缩合体系，DMF为反应溶剂，依次偶联Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，然后经DCM:TFE:AcOH＝7:2:1溶液切割，甲基叔丁基醚沉淀，离心，干燥，再经高效液相制备纯化，获得Fmoc-GGFG-OH。将Fmoc-GGFG-OH和艾日布林置于单口瓶中，加入DMF溶解，降温至0℃，再加入DIPEA(161.3mg,1.25mmol)和DECP(122.3mg,0.75mmol)后于室温反应，HPLC监测，反应完毕后将反应液倒入MTBE(100mL)中，室温搅拌1小时，过滤，滤饼用MTBE洗涤，得到Fmoc-GGFG-Eribulin粗品。将Fmoc-GGFG-Eribulin粗品置于单口瓶中，加入THF，降温至0℃，再加入两当量的LiOH水溶液，于室温反应，HPLC监测，反应完毕后用50％乙酸调pH至6-7左右，旋蒸除去THF，再经高效液相纯化，收集NH2-GGFG-Eribulin纯品。于室温条件下将HATU加入Mal-(PEG)2-COOH和碳酸氢钠的DMF溶液中，搅拌30分钟后，将等当量游离的NH2-GGFG-Eribulin加入上述溶液中再搅拌1小时至TLC检测反应完毕，过滤除去固体，过滤液经HPLC制备即得Mal-(PEG)2-GGFG-Eribulin。
抗HER2互补性双特异ADC的制备方法如下：冰水浴下将ZnCl2(3.0mM)和TCEP(6.0mM)缓慢加入到抗HER2互补性双特异抗体04BS-1123-ST06(1.5mM)溶液中(抗体制剂缓冲液为50mM Histidine-HCl，8％蔗糖，pH 5.5)，其中ZnCl2、TCEP及04BS-1123-ST06抗体的反应终浓度分别为0.10mM、0.20mM和0.05mM，混匀后在8℃震荡还原约16小时。冰水浴下加入溶解于DMSO中的Mal-(PEG)2-VC-PAB-Eribulin或Mal-(PEG)2-GGFG-Eribulin至终浓度0.32mM，于8℃持续反应3小时后加入半胱氨酸至终浓度0.05mM以耗尽过量的Mal-(PEG)2-VC-PAB-Eribulin或Mal-(PEG)2-GGFG-Eribulin。加入EDTA至终浓度0.15mM以螯合Zn2+，使用Pellicon XL Ultrafiltration过滤膜包(Module Ultracel 30kDa/0.005m2，购自Merck)除去相关杂质。最后，使用HIC-HPLC(BioCore HIC-Butyl 5μm/4.6×100mm，购自NanoChrom)分析所制备的ADC的药物-抗体比率(DAR)和异质性，并使用SEC-HPLC(Zenix-C SEC-300，购自赛分科技)分析ADC产物的纯度。
所制备的ADC的平均DAR值可以通过以下公式计算：平均DAR值＝[AUC+1+2(AUC+2)+3(AUC+3)+...+n(AUC+n)]/ΣAUC总]。其中，AUC+1为对应于偶联了一个小分子化合物的ADC的峰曲线下面积，AUC+2为对应于偶联了两个小分子化合物的ADC的峰曲线下面积，以此类推，ΣAUC总为所有峰曲线下面积。本实施例制备的ADC的平均DAR值为4.0±0.5，纯度为＞95％。
5.2抗HER2互补性双特异ADC的抗原结合特异性验证
按照实施例1中第1.4项所述方法，利用ELISA方法检测示例性抗HER2互补性双特异ADC(ST06-VCP-Eribulin)与HER2重组蛋白的结合活性，以及与人源ErbB/HER家族其它成员的交叉结合活性，抗原包括人HER2胞外区重组蛋白以及人EGFR、HER3和HER4胞外区重组蛋白。检测结果如图18所示，抗HER2互补性双特异ADC(ST06-VCP-Eribulin)能够特异性地结合HER2，而与人ErbB/HER家族其它成员无交叉结合活性。所述抗HER2互补性双特异ADC的抗原结合特异性与其相应裸抗04BS-1123-ST06的相当(数据未显示)，表明偶联小分子毒素化合物后并未改变所述互补性双特异抗体的抗原结合特异性。
5.3抗HER2互补性双特异ADC的体外肿瘤细胞杀伤活性的检测
本实施例选用了一系列表达不同水平HER2的肿瘤细胞株来评估所述抗HER2互补性双特异ADC的体外细胞杀伤活性，所用肿瘤细胞株的HER2表达水平以及耐药性情况如表13所示；其中JIMT-1为HER2 过表达且先天型对DS-8201(体外活性)耐药的细胞株，SKOV-3R*为HER2过表达且后天获得型对DS-8201耐药的细胞株。
体外肿瘤细胞杀伤活性检测的具体步骤如下：通过胰酶消化收集各细胞，用含10％ FBS的培养基重悬细胞后接种于96孔白壁板中，每孔细胞数为5,000或10,000。将细胞置于37℃、5％ CO2孵箱中培养，待细胞完全贴壁后，加入梯度稀释于对应培养基中的ST06-VCP-Eribulin、ST06-GGFG-Eribulin或DS-8201。根据各细胞生长速率，在37℃/5％ CO2条件下孵育3～5天后将细胞培养板从孵箱中取出，待平衡至室温后加入CellCount Lite2.0试剂(南京诺唯赞)，振荡混匀2～5分钟使细胞充分裂解；室温静置10分钟后利用多功能酶标仪(Varioskan,Thermo)读取化学发光值。数据用GraphPad Prism 9软件进行分析，并以相对于未处理对照孔的化学发光值的百分比表示。
检测结果如图19和表13所示，在HER2过表达的肿瘤细胞株NCI-N87、SKBR-3及SKOV-3中，所述抗HER2互补性双特异ADC(包括ST06-GGFG-Eribulin和ST06-VCP-Eribulin)和对标分子DS-8201均表现出较强的细胞毒性，并且所述互补性双特异ADC的细胞杀伤活性明显高于DS-8201。在HER2低表达的肿瘤细胞株MDA-MB-361和MDA-MB-453中，所述互补性双特异ADC的细胞杀伤活性显著高于DS-8201；在HER2低表达的肿瘤细胞株ZR-75-1中，所述互补性双特异ADC也显示出有效的细胞杀伤活性，而DS-8201则几乎无细胞杀伤活性。在表达更低水平HER2的肿瘤细胞株T47D、RT-112、MCF-7，以及无HER2表达的肿瘤细胞株MDA-MB-468中，所述互补性双特异ADC则没有明显的细胞毒性，显示所述互补性双特异ADC具有较好的安全性窗口。此外，在HER2高表达且对DS-8201具有先天型/固有耐药性的JIMT-1细胞中，所述互补性双特异ADC(包括ST06-GGFG-Eribulin和ST06-VCP-Eribulin)也表现出显著的细胞毒性。由此，所述抗HER2互补性双特异ADC具有更加广谱的细胞杀伤活性，不仅能够杀伤HER2过表达的肿瘤细胞，也能够直接杀伤HER2低表达的肿瘤细胞，而且对DS-8201不应答的肿瘤细胞也具杀伤作用。
为了进一步验证抗HER2互补性双特异ADC是否能够杀伤对DS-8201治疗产生耐药性或复发/难治(Relapsed/Refractory)的肿瘤细胞，本实施例通过将HER2过表达的SKOV-3细胞在含逐步递增DS-8201浓度的培养基中持续传代培养的方式，筛选获得了对DS-8201具有获得型耐药性(Acquired Resistance)的肿瘤细胞株(命名为SKOV-3R*)，该细胞株能在含有100nM DS-8201的培养基中正常增殖。体外细胞杀伤实验结果显示，所述互补性双特异ADC(包括ST06-GGFG-Eribulin和ST06-VCP-Eribulin)能够有效杀伤后天获得型耐药细胞株SKOV-3R*。
表13.抗HER2互补性双特异ADC及DS-8201的体外细胞杀伤活性

-：无活性。
5.4抗HER2互补性双特异ADC的旁观者杀伤效应
利用HER2高表达细胞株BT474和不表达HER2的细胞株MDA-MB-468对抗HER2互补性双特异ADC ST06-GGFG-Eribulin的旁观者杀伤效应进行了验证，具体方法如下：于第0天将BT474细胞以10,000个细胞/100μL/孔铺入96孔白壁板中，37℃培养过夜；于第1天用培养基稀释ST06-GGFG-Eribulin至20nM，然后作5倍梯度稀释并以100μL/孔加入BT474细胞白壁板中(即起始终浓度为10nM)，置于37℃继续培养；于第3天将MDA-MB-468细胞以10,000个细胞/孔铺入新的96孔白壁板中，37℃培养过夜；于第4天移除MDA-MB-468细胞的培养上清，然后加入100μL/孔取自BT474细胞白壁板中的培养上清(BT474-conditioned Medium)作为旁观者杀伤效应的实验组，并以100μL/孔加入新鲜配制的自10nM起始5倍梯度稀释的ST06-GGFG-Eribulin作为对照组，置于37℃培养；于此同时，利用CellCount Lite2.0 试剂对BT474细胞进行检测。待继续培养72小时后，加入CellCount Lite2.0试剂检测MDA-MB-468细胞的存活情况。利用GraphPad Prism 9软件对实验数据进行分析。结果如图20A所示，ST06-GGFG-Eribulin作用BT474细胞3天后能够造成细胞的显著杀伤，且用其培养上清培养的MDA-MB-468细胞也表现出显著的细胞死亡，而新鲜配制的ADC溶液对该细胞则无任何的杀伤活性，表明ST06-GGFG-Eribulin在杀伤BT474细胞的同时，所释放到培养基中的小分子毒素化合物Eribulin能够杀伤不表达HER2的MDA-MB-468细胞。
此外，本实施例还通过共培养BT474细胞和不表达HER2的Jurkat细胞的方式进一步对ST06-GGFG-Eribulin的旁观者杀伤效应进行了验证，具体方法如下：收集BT474细胞和表达GFP的Jurkat细胞，各自以1×105细胞/孔单独或共同铺入24孔细胞培养板中，随后加入终浓度为1nM的ST06-GGFG-Eribulin，于37℃培养72小时后收集各孔中的细胞至流式管中，离心去上清后用100μL FACS缓冲液(含1％ BSA的PBS)重悬细胞，加入3μL 7-AAD进行死细胞染色后进行流式分析。其中，各管均收集分析10μL细胞液，在排除7-AAD阳性的死细胞后，利用侧向散射光通道和GFP通道对BT474和Jurkat细胞进行区分，最后通过比较在经ST06-GGFG-Eribulin或空白培养基(即，作为对照组，命名为Medium)处理下存活细胞的计数来评估所述互补性双特异ADC是否具有旁观者效应。结果如图20B所示，在单独培养下，ST06-GGFG-Eribulin对BT474细胞显示出明显的杀伤活性，而对Jurkat细胞则无杀伤，但在两种细胞共培养下，ST06-GGFG-Eribulin对BT474和Jurkat细胞都造成了明显杀伤。
综上，以上两个实验结果均表明ST06-GGFG-Eribulin具有旁观者杀伤效应。
实施例6抗HER2互补性双特异ADC在小鼠皮下异种移植模型中的抗肿瘤活性检测
6.1抗HER2互补性双特异ADC在基于肿瘤细胞株构建的小鼠皮下异种移植模型中的抑瘤活性
本实施例检测了抗HER2互补性双特异ADC在3种基于人肿瘤细胞株所构建的小鼠皮下异种移植肿瘤模型中的抗肿瘤活性，其中参比对照药物为DS-8201(商品名Enhertu，购自第一三共制药)；所述3种肿瘤模型为：HER2过表达的NCI-N87胃癌肿瘤模型、HER2中度表达的JIMT-1乳腺癌肿瘤模型、和HER2低表达的RT-112膀胱癌肿瘤模型。具体方法如下：待细胞株(NCI-N87、JIMT-1、及RT-112)培养至对数生长期后，收集细胞并以每只5～10×106个细胞接种至免疫缺陷型小鼠(Nude或SCID)的右侧前肢背部皮下。待肿瘤生长至200±50mm3后将小鼠随机分组(每组8只荷瘤小鼠)，包括ST06-GGFG-Eribulin试验组、DS-8201参比对照组、溶媒对照组(含8％蔗糖的50mM Histidine-HCl溶液，pH 5.5)、以及抗体-小分子药物混合物ADMix对照组(ADMix由裸抗04BS-1123-ST06和Eribulin以摩尔比1:4混合得到)。所有荷瘤小鼠均经尾静脉注射给药，不同模型中各组的给药剂量和给药频率如图21所示。给药后每周测量两次并记录小鼠的体重和肿瘤尺寸(长径、短径)，并以下列公式计算肿瘤体积：肿瘤体积＝(长径)×(短径)2×0.5。
实验结果如图21所示，与溶媒对照相比，DS-8201和ST06-GGFG-Eribulin在NCI-N87(图21A)、JIMT-1(图21B)和RT-112(图21C)肿瘤模型中均表现出显著的肿瘤生长抑制作用，而且ST06-GGFG-Eribulin的抗肿瘤活性在NCI-N87和JIMT-1模型中均表现出剂量依赖性的趋势。在相同的给药剂量(3mg/kg)下，ST06-GGFG-Eribulin在HER2过表达的NCI-N87肿瘤模型中表现出与DS-8201相当的抗肿瘤活性，但在HER2中等表达的JIMT-1肿瘤模型中则表现出比DS-8201更强的抗肿瘤活性，且具有显著的统计学差异(p＜0.01)。在HER2低表达的RT-112肿瘤模型中，考虑到药物分子量及DAR的差异，ST06-GGFG-Eribulin在等当量DAR的给药剂量(9mg/kg)下表现出明显超越DS-8201的抗肿瘤活性，且所述抗肿瘤活性的差异具有显著的统计学意义(p＜0.01)。整个实验期间，未观察到荷瘤小鼠的体重下降或其它毒副反应(数据未显示)。
6.2抗HER2互补性双特异ADC在对DS-8201具有后天获得型耐药性的小鼠皮下异种移植瘤模型中的抑瘤活性
为了进一步验证所述抗HER2互补性双特异ADC对经DS-8201治疗后产生耐药性或复发的肿瘤是否具有杀伤作用，本实施例利用NCI-N87肿瘤细胞株构建了对DS-8201具有后天获得型耐药性的小鼠异种移植肿瘤模型。构建方法如下：将野生型NCI-N87细胞在含逐步递增DS-8201浓度的培养基中持续传代培养，筛选获得对DS-8201具有一定耐药性的细胞株亚群并命名其为NCI-N87(R)。将NCI-N87(R)细胞在含10％血清的RPMI-1640培养基中进行扩增培养至指数生长期，收集细胞并重悬于PBS与Matrigel的混合液(体积比为1:1)中，然后于每只裸鼠(BALB/c Nude)的右侧前肢背部皮下接种1×107个细胞，每次接种5只雌性裸鼠，并开展获得型耐药性肿瘤的体内筛选，具体筛选步骤如下：待小鼠肿瘤体积达到200±50mm3时，尾静脉注射DS-8201(剂量为3mg/kg)，每周给药一次以筛选肿瘤在给药后仍稳定生长的荷瘤裸鼠；给药次数为3～5次，取决于肿瘤的生长速率。在肿瘤体积增长至900～1000mm3后，将小鼠安乐死并收集肿瘤，在剔除坏死组织后将肿瘤切割成约30mm3的小块，接种于5只雌性裸鼠背部右侧近前肢背部皮下并开展下一轮的DS-8201给药和筛选。经过如此3轮的筛选后，发现5只荷瘤裸鼠的肿瘤在DS-8201(剂量为3mg/kg)给药后均能稳定生长，且增长趋势较为一致。待肿瘤体积增长至约1000mm3后，随机选取 2只小鼠安乐死，收集肿瘤并剔除坏死组织，切割成约30mm3的小块后接种于50只雌性裸鼠背部右侧近前肢背部皮下。
在上述50只荷瘤小鼠中，选择肿瘤长势较为均一且平均体积达到约210mm3的29只小鼠进行随机分组，包括2个ST06-GGFG-Eribulin试验组(按照给药剂量分为3mg/kg组和10mg/kg组)、1个DS-8201对照组(给药剂量为3mg/kg)和1个溶媒对照组，其中给药剂量为3mg/kg的ST06-GGFG-Eribulin试验组、DS-8201对照组和溶媒对照组各8只小鼠，给药剂量为10mg/kg的ST06-GGFG-Eribulin试验组为5只小鼠。各组小鼠通过尾静脉注射给药，共给药4周，其中DS-8201组和溶媒组每周给药一次，ST06-GGFG-Eribulin试验组在第一周给药一次后，余下的3周中每周给药2次。小鼠每周进行两次测量并记录各组小鼠体重和肿瘤的尺寸，并以下列公式计算肿瘤体积：肿瘤体积＝(长径)×(短径)2×0.5。
实验结果如图22所示，小鼠肿瘤在DS-8201给药后持续稳定生长，表明建模成功，即肿瘤模型确为对DS-8201具有后天获得型耐药性的异种移植肿瘤模型。ST06-GGFG-Eribulin在3mg/kg和10mg/kg的剂量下均显示出抗肿瘤活性；尤其是在10mg/kg的剂量(等当量DAR)下，ST06-GGFG-Eribulin给药后肿瘤几乎完全消退，显示所述互补性双特异ADC(ST06-GGFG-Eribulin)能够突破DS-8201的获得型耐药性，对DS-8201复发难治性(Relapsed/Refractory)肿瘤具有潜在疗效。整个实验期间，未观察到荷瘤小鼠的体重下降或其它毒副反应(数据未显示)。

Claims (40)

  1. 一种分离的抗HER2抗体或其抗原结合片段，其中，所述抗HER2抗体或其抗原结合片段具有以下特性中的至少一项：
    (1)所述抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域1、3和/或4；
    (2)所述抗HER2抗体或其抗原结合片段不影响HER2及其介导的下游信号转导通路，包括不诱导、不阻断、不抑制HER2-酪氨酸残基磷酸化和/或去磷酸化，和/或不诱导、不阻断、不抑制依赖于或不依赖于配体诱导的HER2二聚化及其下游信号转导通路，所述HER2二聚化包括HER2/HER4二聚化和HER2/HER3二聚化，所述配体包括NRG-1和Heregulins；以及
    (3)所述抗HER2抗体或其抗原结合片段不与人源ErbB/HER家族其它成员(包括EGFR、HER3和HER4)发生交叉结合反应。
  2. 如权利要求1所述的抗HER2抗体或其抗原结合片段，其中，所述抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域3，其包含：(1)氨基酸序列为X1YGMS(SEQ ID NO:217)的HCDR1，X1＝S、N和D；(2)氨基酸序列为SISGX2GX3YX4KYX5X6X7VKG(SEQ ID NO:218)的HCDR2，X2＝G或S；X3＝S或N；X4＝T或A；X5＝P、A、G或V；X6＝D、G、E、P、Q或R；X7＝S、K或N；(3)氨基酸序列为DYX8GFFDV(SEQ ID NO:219)的HCDR3，X8＝A、I、N、R、S或V；(4)氨基酸序列为RSSQSLX9X10SNX11NTYLH(SEQ ID NO:220)的LCDR1，X9＝V或L；X10＝H或S；X11＝G、A、I、S、R或T；(5)氨基酸序列为KVSNRX12S(SEQ ID NO:221)的LCDR2，X12＝F、D或P；(6)氨基酸序列为X13QSTHVPX14T(SEQ ID NO:222)的LCDR3，X13＝S或Q；X14＝Y或W，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  3. 如权利要求1或2所述的抗HER2抗体或其抗原结合片段，其包含：(1)如SEQ ID NO:11、12、或13所示的HCDR1；(2)如SEQ ID NO:14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、或31所示的HCDR2；和(3)如SEQ ID NO:32、33、34、35、36、或37所示的HCDR3；以及(4)如SEQ ID NO:38、39、40、41、42、43、44、或45所示的LCDR1；(5)如SEQ ID NO:46、47、或48所示的LCDR2；和(6)如SEQ ID NO:49、50、或51所示的LCDR3；
    进一步，所述的抗HER2抗体或其抗原结合片段包含：
    (1)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14、15、16、17、18、19、或20所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:38所示的LCDR1，SEQ ID NO:46(其中X12＝F)所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (2)如SEQ ID NO:12或13所示的HCDR1，SEQ ID NO:14所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:38所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (3)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:38所示的LCDR1，SEQ ID NO:47或48所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (4)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:39、40、41、42、43、44、或45所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (5)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:38所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:50或51所示的LCDR3；或
    (6)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14所示的HCDR2，SEQ ID NO:33、34、35、36、或37所示的HCDR3，以及SEQ ID NO:38所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (7)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:14、21、22、23、24、25、26、27、28、29、30、或31所示的HCDR2，SEQ ID NO:37所示的HCDR3，以及SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (8)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:21所示的HCDR2，SEQ ID NO:32所示的HCDR3，以及SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (9)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:21、23、24、27、28、29、30、或31所示的HCDR2，SEQ ID NO:37所示的HCDR3，以及SEQ ID NO:42所示的LCDR1、SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3；或
    (10)如SEQ ID NO:11所示的HCDR1，SEQ ID NO:24、29、30、或31所示的HCDR2，SEQ ID  NO:37所示的HCDR3，以及SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2，SEQ ID NO:49所示的LCDR3。
  4. 如权利要求1所述的抗HER2抗体或其抗原结合片段，其中，所述抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域1，其包含：(1)氨基酸序列为DYX15MH(SEQ ID NO:223)的HCDR1，X15＝S或A；(2)氨基酸序列为WINTX16TGX17PTYADX18X19KG(SEQ ID NO:224)的HCDR2，X16＝E、N、G、Y或I；X17＝E、D或S；X18＝D、K或N；X19＝F或V；(3)氨基酸序列为VGX20X21X22YAMDY(SEQ ID NO:225)的HCDR3，X20＝R或Y；X21＝Y或G；X22＝D或S；(4)氨基酸序列为X23ASQDVYTAVA(SEQ ID NO:226)的LCDR1，X23＝K或R；(5)氨基酸序列为X24ASX25RX26T(SEQ ID NO:227)的LCDR2，X24＝S、A、D、E、K、L、Q、R、W或Y；X25＝Y、D、E、K、N、Q、S或T；X26＝Y、A、E、P或Q；(6)氨基酸序列为QQX27YSTPPT(SEQ ID NO:228)的LCDR3，X27＝H、S、A或Y，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  5. 如权利要求1或4所述的抗HER2抗体或其抗原结合片段，其包含：如SEQ ID NO:98或99所示的HCDR1；如SEQ ID NO:100、101、102、103、104、105、106、107、108、或109所示的HCDR2；如SEQ ID NO:110、111、112、或113所示的HCDR3；以及如SEQ ID NO:114或115所示的LCDR1；如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、或138所示的LCDR2；如SEQ ID NO:139、140、141、或142所示的LCDR3；
    进一步，所述的抗HER2抗体或其抗原结合片段包含：
    (1)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:100所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：114所示的LCDR1，SEQ ID NO:116、117、118、119、120、121、122、123、124、125、126、127、或128所示的LCDR2，SEQ ID NO:139所示的LCDR3；或
    (2)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:100所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：114所示的LCDR1，SEQ ID NO:116所示的LCDR2，SEQ ID NO:140、141、或142所示的LCDR3；或
    (3)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:101、102、103、104、105、106、107、108、或109所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：114所示的LCDR1，SEQ ID NO:116所示的LCDR2，SEQ ID NO:139所示的LCDR3；或
    (4)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:100所示的HCDR2，SEQ ID NO:111、112、或113所示的HCDR3，SEQ ID NO：114所示的LCDR1，SEQ ID NO:116所示的LCDR2，SEQ ID NO:139所示的LCDR3；或
    (5)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:100所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：115所示的LCDR1，SEQ ID NO:116所示的LCDR2，SEQ ID NO:139所示的LCDR3；或
    (6)如SEQ ID NO:99所示的HCDR1，SEQ ID NO:100所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：114所示的LCDR1，SEQ ID NO:116所示的LCDR2，SEQ ID NO:139所示的LCDR3；或
    (7)如SEQ ID NO:98所示的HCDR1，SEQ ID NO:102所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：115所示的LCDR1，SEQ ID NO:129、130、131、132、133、134、135、136、137、或138所示的LCDR2氨基酸序列，SEQ ID NO:142所示的LCDR3；
    优选地，所述抗HER2抗体或其抗原结合片段包含：如SEQ ID NO:98所示的HCDR1，SEQ ID NO:102所示的HCDR2，SEQ ID NO:110所示的HCDR3，SEQ ID NO：115所示的LCDR1，SEQ ID NO:129、133、136、或138所示的LCDR2，SEQ ID NO:142所示的LCDR3。
  6. 如权利要求1所述的抗HER2抗体或其抗原结合片段，其中，所述的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域4，其包含：如SEQ ID NO:199所示的HCDR1，SEQ ID NO:200所示的HCDR2，SEQ ID NO:201所示的HCDR3，以及SEQ ID NO:202所示的LCDR1，SEQ ID NO:203所示的LCDR2和SEQ ID NO:204所示的LCDR3；或者，
    所述的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域1，其包含：如SEQ ID NO:205所示的HCDR1，SEQ ID NO:206所示的HCDR2，SEQ ID NO:207所示的HCDR3，以及SEQ ID NO:208所示的LCDR1，SEQ ID NO:209所示的LCDR2和SEQ ID NO:210所示的LCDR3；或者；
    所述的抗HER2抗体或其抗原结合片段能够特异性地结合HER2胞外区亚结构域1，其包含：如SEQ ID NO:211所示的HCDR1，SEQ ID NO:212所示的HCDR2，SEQ ID NO:213所示的HCDR3，以及SEQ ID  NO:214所示的LCDR1，SEQ ID NO:215所示的LCDR2和SEQ ID NO:216所示的LCDR3。
  7. 如权利要求1-3中任一项所述的抗HER2抗体或其抗原结合片段，其包含：与SEQ ID NO:1、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、或80所示的氨基酸序列具有至少80％同一性的重链可变区VH，以及与SEQ ID NO:2、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、或97所示的氨基酸序列具有至少80％同一性的轻链可变区VL；
    进一步，所述的抗HER2抗体或其抗原结合片段包含：
    (1)分别与SEQ ID NO:1所示的VH和SEQ ID NO:2所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或
    (2)分别与SEQ ID NO:52所示的VH和SEQ ID NO:81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、或97所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或
    (3)分别与SEQ ID NO:53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、或68所示的VH和SEQ ID NO:81所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或
    (4)分别与SEQ ID NO:68、69、70、71、72、73、74、75、76、77、78、79、或80所示的VH和SEQ ID NO:92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；
    优选地，所述的抗HER2抗体或其抗原结合片段包含分别与SEQ ID NO:73、78、79、或80所示的VH和SEQ ID NO:92所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  8. 如权利要求1、4或5所述的抗HER2抗体或其抗原结合片段，其包含：与SEQ ID NO:3、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、或170所示的氨基酸序列具有至少80％同一性的重链可变区VH，以及与SEQ ID NO:4、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、或198所示的氨基酸序列具有至少80％同一性的轻链可变区VL；
    进一步，所述的抗HER2抗体或其抗原结合片段包含：
    (1)分别与SEQ ID NO:3所示的VH和SEQ ID NO:4所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者
    (2)分别与SEQ ID NO:143所示的VH和SEQ ID NO:171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、或188所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者
    (3)分别与SEQ ID NO:144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、或170所示的VH和SEQ ID NO:171所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者
    (4)分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；
    优选地，所述的抗HER2抗体或其抗原结合片段包含分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；
    更优选地，所述的抗HER2抗体或其抗原结合片段包含分别与SEQ ID NO:169所示的VH和SEQ ID NO:189、193、196、或198所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  9. 如权利要求1或6所述的抗HER2抗体或其抗原结合片段，其包含重链可变区VH和轻链可变区VL，所述重链可变区VH和轻链可变区VL包含：
    (1)分别与SEQ ID NO:5所示的VH和SEQ ID NO:6所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者，
    (2)分别与SEQ ID NO:7所示的VH和SEQ ID NO:8所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；或者，
    (3)分别与SEQ ID NO:9所示的VH和SEQ ID NO:10所示的VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  10. 一种抗HER2互补性双特异抗体，其包含两种能够非竞争性地同时结合HER2胞外区的抗原结合结构域，其中第一抗原结合结构域和第二抗原结合结构域各自特异性地结合HER2胞外区的不同表位，所述表位位于HER2胞外区亚结构域1、3和4中，所述互补性双特异抗体包含以下特性中的至少一项：
    (1)所述互补性双特异抗体能够交联/聚集肿瘤细胞表面的HER2，进而引发HER2交联体的快速、高效内吞，表现为在HER2低表达肿瘤细胞中的内吞率高达60％以上，在HER2过表达肿瘤细胞中的内吞率高达80％以上；
    (2)所述互补性双特异抗体能够有效地引发肿瘤细胞表面HER2的溶酶体转运和降解，显著降低细胞表面HER2的表达，以致能显著抑制HER2过表达肿瘤细胞的增殖；
    (3)所述互补性双特异抗体不影响HER2及其介导的下游信号转导通路，包括不诱导、不阻断、不抑制配体诱导的HER2二聚化及其下游信号转导通路的激活，因此所述互补性双特异抗体不影响正常组织/细胞、包括心肌细胞中HER2及其介导的下游信号转导通路的功能与调控，所述配体包括NRG-1；
    (4)所述互补性双特异抗体能够特异性地结合HER2，而不与人源ErbB/HER家族其它成员(包括EGFR、HER3和HER4)交叉结合。
  11. 如权利要求10所述的互补性双特异抗体，其中，所述第一抗原结合结构域和第二抗原结合结构域中的一个特异性地结合HER2胞外区亚结构域1，另一个特异性地结合HER2胞外区亚结构域3；
    进一步，所述第一抗原结合结构域包含：(1)氨基酸序列为X1YGMS的HCDR1，X1＝S、N和D；(2)氨基酸序列为SISGX2GX3YX4KYX5X6X7VKG的HCDR2，X2＝G或S；X3＝S或N；X4＝T或A；X5＝P、A、G或V；X6＝D、G、E、P、Q或R；X7＝S、K或N；(3)氨基酸序列为DYX8GFFDV的HCDR3，X8＝A、I、N、R、S或V；(4)氨基酸序列为RSSQSLX9X10SNX11NTYLH的LCDR1，X9＝V或L；X10＝H或S；X11＝G、A、I、S、R或T；(5)氨基酸序列为KVSNRX12S的LCDR2，X12＝F、D或P；(6)氨基酸序列为X13QSTHVPX14T的LCDR3，X13＝S或Q；X14＝Y或W，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；
    所述第二抗原结合结构域包含：(1)氨基酸序列为DYX15MH的HCDR1，X15＝S或A；(2)氨基酸序列为WINTX16TGX17PTYADX18X19KG的HCDR2，X16＝E、N、G、Y或I；X17＝E、D或S；X18＝D、K或N；X19＝F或V；(3)氨基酸序列为VGX20X21X22YAMDY的HCDR3，X20＝R或Y；X21＝Y或G；X22＝D或S；(4)氨基酸序列为X23ASQDVYTAVA的LCDR1，X23＝K或R；(5)氨基酸序列为X24ASX25RX26T的LCDR2，X24＝S、A、D、E、K、L、Q、R、W或Y；X25＝Y、D、E、K、N、Q、S或T；X26＝Y、A、E、P或Q；(6)氨基酸序列为QQX27YSTPPT的LCDR3，X27＝H、S、A或Y，或者分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  12. 如权利要求10或11所述的抗HER2互补性双特异抗体，其中，所述第一抗原结合结构域包含：如SEQ ID NO:11所示的HCDR1，SEQ ID NO:21、23、24、27、28、29、30、或31所示的HCDR2，SEQ ID NO:37所示的HCDR3，以及SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2和SEQ ID NO:49所示的LCDR3；
    所述第二抗原结合结构域包含：如SEQ ID NO:98所示的HCDR1，SEQ ID NO:102所示的HCDR2，SEQ ID NO:110所示的HCDR3，以及SEQ ID NO:115所示的LCDR1，SEQ ID NO:129、130、131、132、133、134、135、136、137、或138所示的LCDR2和SEQ ID NO:142所示的LCDR3；
    进一步，所述第一抗原结合结构域包含如SEQ ID NO:11所示的HCDR1，SEQ ID NO:24、29、30、或31所示的HCDR2，SEQ ID NO:37所示的HCDR3，以及SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2和SEQ ID NO:49所示的LCDR3；
    所述第二抗原结合结构域包含：如SEQ ID NO:98所示的HCDR1，SEQ ID NO:102所示的HCDR2，SEQ ID NO:110所示的HCDR3，以及SEQ ID NO:115所示的LCDR1，SEQ ID NO:133所示的LCDR2和SEQ ID NO:142所示的LCDR3。
  13. 如权利要求10或11所述的抗HER2互补性双特异抗体，其中，所述第一抗原结合结构域包含：如SEQ ID NO:70、72、73、76、77、78、79、或80所示的VH，以及如SEQ ID NO:92所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、 97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域包含：如SEQ ID NO:169所示的VH，以及SEQ ID NO:189、190、191、192、193、194、195、196、197、或198所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；
    优选地，所述第一抗原结合结构域包含：如SEQ ID NO:73、78、79、或80所示的VH，以及SEQ ID NO:92所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域包含：如SEQ ID NO:169所示的VH，以及SEQ ID NO:193所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  14. 如权利要求10-12中任一项所述的抗HER2互补性双特异抗体，所述双特异抗体的结构为DVD-Ig形式，其中，所述第一抗原结合结构域或第二抗原结合结构域中的一个为Fv结构域，且另一个为Fab或IgG结构域，所述Fv结构域通过连接子与所述Fab或IgG结构域连接；
    进一步，所述第一抗原结合结构域特异性地结合HER2胞外区亚结构域3，且为Fv结构域，所述第二抗原结合结构域特异性地结合HER2胞区亚外结构域1，且为Fab结构域或IgG结构域，所述第一或第二抗原结合结构域包含重链可变区VH和轻链可变区VL，所述Fv结构域中的VH结构域C-末端通过连接子与所述Fab或IgG结构域中的VH结构域N-末端连接，所述Fv结构域中的VL结构域C-末端通过连接子与所述Fab或IgG结构域中的VL结构域N-末端连接。
  15. 如权利要求10-14中任一项所述的抗HER2互补性双特异抗体，包含4条多肽链：
    (1)其中两条多肽链包含VH1-L1-VH2-C-(Fc)n，其中VH1表示特异性结合HER2胞外区亚结构域3的第一抗原结合结构域的重链可变区，L1表示连接子，VH2表示特异性结合HER2胞外区亚结构域1的第二抗原结合结构域的重链可变区，C表示重链恒定区CH1，Fc表示重链恒定区Fc结构域，n是0或1；以及
    (2)另外两条多肽链包含VL1-L2-VL2-CL，其中VL1表示特异性结合HER2胞外区亚结构域3的第一抗原结合结构域的轻链可变区，L2表示连接子，VL2表示特异性结合HER2胞外区亚结构域1的第二抗原结合结构域的轻链可变区，CL是IgG轻链恒定区；
    所述L1和L2连接子包含(G4S)n序列，n代表G4S的拷贝数，其为大于0的整数，L1和L2连接子的拷贝数n可以相同或不同。
  16. 如权利要求15所述的抗HER2互补性双特异抗体，其中，所述VH1包含SEQ ID NO:11所示的HCDR1，SEQ ID NO:24、29、30、或31所示的HCDR2和SEQ ID NO:37所示的HCDR3；VH2包含SEQ ID NO:98所示的HCDR1，SEQ ID NO:102所示的HCDR2和SEQ ID NO:110所示的HCDR3；所述VL1包含SEQ ID NO:42所示的LCDR1，SEQ ID NO:46所示的LCDR2和SEQ ID NO:49所示的LCDR3；VL2包含SEQ ID NO:115所示的LCDR1，SEQ ID NO:133所示的LCDR2和SEQ ID NO:142所示的LCDR3。
  17. 如权利要求15或16所述的抗HER2互补性双特异抗体，其中，所述VH1包含如SEQ ID NO:73、78、79、或80所示的VH，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述VH2包含如SEQ ID NO:169所示的VH，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述VL1包含如SEQ ID NO:92所示的VL，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述VL2包含如SEQ ID NO:193所示的VL，或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  18. 如权利要求15-17中任一项所述的抗HER2互补性双特异抗体，其中，所述L1包含SEQ ID NO:229所示的连接子氨基酸序列；所述L2包含SEQ ID NO:230所示的连接子氨基酸序列；所述Fc包含工程化的Fc结构域，优选地，包括如SEQ ID NO:232所示的具有L234F/L235E/P331S(EU编号系统)氨基酸取代的人IgG1 Fc的氨基酸序列；所述CL选自人κ恒定区或人λ恒定区，优选地，如SEQ ID NO:233所示的人κ恒定区氨基酸序列。
  19. 一种抗HER2互补性双特异抗体-药物偶联物(ADC)，包含如权利要求10-18中任一项所述的抗HER2互补性双特异抗体、小分子毒素化合物和可裂解接头，所述ADC包含以下特性中的至少一项：
    (1)所述ADC能够同时结合肿瘤细胞表面HER2的两个不同表位，交联HER2成簇，以及引发ADC的快速内吞和溶酶体转运，因此显著提高了ADC中的小分子毒素化合物进入靶细胞及其在靶细胞内释放 的效率，使得所述ADC具有更加广谱的杀伤活性，表现为不仅在HER2过表达的肿瘤细胞中具有比现有的HER2靶向药物更强的杀伤活性，而且在HER2低表达的肿瘤细胞中也能发挥直接杀伤作用，所述现有的HER2靶向药物包括Trastuzumab、Pertuzumab、T-DM1和DS-8201；
    (2)所述ADC对上述现有的HER2靶向药物产生耐药性或复发的肿瘤具有杀伤作用；
    (3)所述ADC不影响HER2的生物学活性及其介导的信号转导通路的调控，不会干扰心肌细胞中HER2的正常功能，且不具有Fc效应子功能，因此其临床安全性显著高于上述现有的HER2靶向药物；
    进一步，所述的ADC由式(Ⅰ)表示：
    Ab-(L-D)p  (Ⅰ)
    其中，Ab表示如权利要求10-18中任一项所述的抗HER2互补性双特异抗体，所述Ab包含第一和第二抗原结合结构域，所述第一抗原结合结构域特异性地结合HER2胞外区亚结构域3，且为Fv结构域，所述的第二抗原结合结构域特异性地结合HER2胞外区亚结构域1，且为IgG结构域，所述的第一或第二抗原结合结构域包含重链可变区和轻链可变区；
    D表示小分子毒素化合物；
    L表示可裂解接头；以及
    p为2至8。
  20. 如权利要求19所述的ADC，其中，所述第一抗原结合结构域包含：(1)如SEQ ID NO:11所示的HCDR1，(2)如SEQ ID NO:24、29、30、或31所示的HCDR2，(3)如SEQ ID NO:37所示的HCDR3，(4)如SEQ ID NO:42所示的LCDR1，(5)如SEQ ID NO:46所示的LCDR2，以及(6)如SEQ ID NO:49所示的LCDR3；所述第二抗原结合结构域包含：(1)如SEQ ID NO:98所示的HCDR1，(2)如SEQ ID NO:102所示的HCDR2，(3)如SEQ ID NO:110所示的HCDR3，(4)如SEQ ID NO:115所示的LCDR1，(5)如SEQ ID NO:133所示的LCDR2，以及(6)如SEQ ID NO:142所示的LCDR3。
  21. 如权利要求19或20所述的ADC，其中，所述第一抗原结合结构域包含如SEQ ID NO:73、78、79、或80所示的VH和如SEQ ID NO:92所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列；所述第二抗原结合结构域包含如SEQ ID NO:169所示的VH和如SEQ ID NO:193所示的VL，或分别与所述的VH和VL具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％同一性的氨基酸序列。
  22. 如权利要求19-21中任一项所述的ADC，其中，所述Ab进一步包含恒定区，所述恒定区包括重链恒定区和轻链恒定区，所述重链恒定区包含如SEQ ID NO:232所示的人IgG1 Fc，所述轻链恒定区包含如SEQ ID NO:233所示的人κ恒定区。
  23. 如权利要求19-22中任一项所述的ADC，其中，所述小分子毒素化合物包括艾日布林、奥瑞他汀类衍生物、Tubulysins、Cryptomycins、美登素类衍生物、拓扑异构酶抑制剂、吡咯并苯并二氮杂卓(PBD)、卡奇霉素及其衍生物、倍癌霉素、烷基化疗剂以及具有烷基化形式的其他化合物、抗代谢物、有丝分裂抑制剂、和细胞毒性抗生素；
    进一步，所述奥瑞他汀类衍生物包括MMAE、MMAF、MMAD；所述美登素类衍生物包括DM1、DM2、DM3、DM4；所述拓扑异构酶抑制剂包括喜树碱类衍生物SN-38、依沙替康和DXd；所述烷基化疗剂以及具有烷基化形式的其他化合物包括氮芥、亚乙基亚胺化合物、烷基磺酸盐、亚硝基脲、顺铂、达卡巴嗪；所述抗代谢物包括叶酸、嘌呤或嘧啶拮抗剂；所述有丝分裂抑制剂包括长春花生物碱和鬼臼毒素的衍生物；所述细胞毒性抗生素包括蒽环类抗生物、放线菌素、博来霉素；优选的小分子毒素化合物是艾日布林。
  24. 如权利要求19-23中任一项所述的ADC，其中，所述的可裂解接头包含可裂解肽部分和至少一种间隔区；
    进一步，所述可裂解肽部分包含氨基酸单元，所述氨基酸单元包含由组织蛋白酶B裂解的Phe-Lys、Val-Cit(VC)、Glu-Val-Cit、或Gly-Gly-Phe-Gly(GGFG)氨基酸序列；
    所述间隔区包含与抗体缀合的间隔区，和/或与小分子毒素化合物缀合的第二间隔区，所述与抗体缀合的间隔区是亲水性间隔区，其包含一个或多个聚乙二醇(PEG)，所述第二间隔区包含对氨基苯甲基单元。
  25. 如权利要求24所述的ADC，所述的氨基酸单元包含VC和GGFG；
    所述的亲水性间隔区包含2个PEG，所述的亲水性间隔区与顺丁烯二酰亚胺(Mal)构成Mal-间隔区而附接于抗体；所述第二间隔区包含对氨基苯甲基羰基(PAB)。
  26. 如权利要求24或25所述的ADC，所述的可裂解接头包含Mal-间隔区和可裂解肽部分，所述Mal-间隔区包含2个PEG，所述可裂解肽部分包含二肽VC和四肽GGFG；或者所述的可裂解接头包含Mal-间隔区、可裂解肽部分和第二间隔区，所述Mal-间隔区包含2个PEG，所述可裂解肽部分包含二肽VC，所述第二间隔区包含PAB。
  27. 如权利要求24-26中任一项所述的ADC，所述的可裂解接头包含Mal-(PEG)2-VC，Mal-(PEG)2-GGFG，或Mal-(PEG)2-VC-PAB。
  28. 如权利要求24-27中任一项所述的ADC，其中所述可裂解接头的可裂解肽部分可以直接或通过第二间隔区偶联至所述小分子毒素化合物，以制备为包含药物有效载荷的ADC，所述小分子毒素化合物是艾日布林。
  29. 如权利要求28所述的ADC，所述可裂解接头包含Mal-(PEG)2-GGFG，所述药物有效载荷包含所述可裂解接头的可裂解肽部分GGFG通过羧基偶联至艾日布林C-35胺而获得的化合物Mal-(PEG)2-GGFG-Eribulin；或者，
    所述可裂解接头包含Mal-(PEG)2-VC-PAB，所述药物有效载荷包含所述可裂解接头通过第二间隔区PAB偶联至艾日布林的C-35胺而获得的化合物Mal-(PEG)2-VC-PAB-Eribulin。
  30. 如权利要求19-29中任一项所述的ADC，所述p为4至8。
  31. 一种核酸，其编码如权利要求1-9中任一项所述的抗HER2抗体或其抗原结合片段、权利要求10-18中任一项所述的抗HER2互补性双特异抗体、或权利要求19-30中任一项所述的ADC中抗体部分的核酸。
  32. 一种表达载体，其能够表达权利要求31所述的核酸。
  33. 一种宿主细胞，包含权利要求31所述的核酸或权利要求32所述的表达载体。
  34. 一种使用权利要求33所述的宿主细胞制备权利要求1-9中任一项所述的抗HER2抗体或其抗原结合片段、权利要求10-18中任一项所述的抗HER2互补性双特异抗体、或权利要求19-30任一项所述的ADC中抗体部分的方法，包括：(i)在所述的宿主细胞中表达所述抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或所述ADC中抗体部分，和(ii)从所述的宿主细胞或细胞培养物中分离所述抗HER2抗体或其抗原结合片段、抗HER2互补性双特异抗体、或所述ADC中抗体部分。
  35. 一种制备如权利要求19-30中任一项所述的ADC的方法，包括：所述小分子毒素化合物的亲核基团或亲电子基团与可裂解接头反应，经由共价键形成药物有效载荷，再与抗体的亲核基团或亲电子基团的反应，或者优选地，所述抗体铰链区的链间二硫键经还原后产生的游离半胱氨酸残基与药物有效载荷的反应官能团反应形成共价键，所述反应官能团包括顺丁烯二酰亚胺基。
  36. 一种药物组合物，包含如权利要求1-9中任一项所述的抗HER2抗体或其抗原结合片段、权利要求10-18中任一项所述的抗HER2互补性双特异抗体、或权利要求19-30任一项所述的ADC，以及药学上可接受的载体。
  37. 一种药盒，包含有效量的如权利要求1-9中任一项所述的抗HER2抗体或其抗原结合片段、权利要求10-18中任一项所述的抗HER2互补性双特异抗体、权利要求19-30任一项所述的抗HER2互补性双特异ADC、或权利要求36所述的药物组合物，以及任选的至少一种另外的肿瘤治疗剂；
    进一步，所述另外的肿瘤治疗剂包括HER2拮抗剂、EGFR拮抗剂、HER3拮抗剂、MET拮抗剂、IGF1R拮抗剂、B-Raf抑制剂、PDGFR-α抑制剂、PDGFR-β抑制剂、PDGF配体抑制剂、VEGF拮抗剂、VEGF受体激酶抑制剂、DLL4拮抗剂、Ang2拮抗剂、FOLH1拮抗剂、STEAP1或STEAP2拮抗剂、TMPRSS2拮抗剂、MSLN拮抗剂、MUC16拮抗剂、CLEC12A拮抗剂、PD-1或PD-L1阻断剂、激素受体调节剂、芳香酶抑制剂、激酶抑制剂、细胞因子激动剂或细胞因子抑制剂、化学治疗剂。
  38. 一种治疗表达HER2的癌症的方法，包括向有需要的受试者施用有效量的如权利要求10-18中任一项所述的抗HER2互补性双特异抗体、权利要求19-30中任一项所述的抗HER2互补性双特异ADC、权利要求36所述的药物组合物、或权利要求37所述的药盒。
  39. 如权利要求38所述的方法，其中，所述癌症包括乳腺癌、卵巢癌、宫颈癌、结直肠癌、胃癌、食管癌、肺癌、头颈癌、黑色素瘤、胰腺癌、肝癌、胆管癌、肾癌、膀胱癌、甲状腺癌、前列腺癌和子宫内膜癌，所述癌症还包括各阶段的癌症，例如早期癌症、非转移性癌症、原发性癌症、晚期癌症、局部晚期癌症、转移性癌症或缓解中的癌症；所述癌症还包括HER2过表达的癌症、HER2表达水平相对较低的癌症、对现有的HER2靶向治疗剂不响应或响应较差的癌症、和/或对现有HER2靶向治疗剂产生耐药性或复发的癌症，其中所述现有的HER2靶向治疗剂包括Trastuzumab、Pertuzumab、T-DM1和DS-8201；
    所述的受试者可以是人、非人灵长类动物或其它哺乳动物如狗、小鼠、大鼠；所述受试者进一步包括不适合接受现有HER2靶向疗法或难治疗、或在接受现有的HER2靶向疗法后产生耐药性或复发的患者。
  40. 一种检测和/或测量样品中HER2或表达HER2的肿瘤细胞的方法，以及筛选对如权利要求19-30中任一项所述的ADC治疗有响应的癌症患者的方法，包括将如权利要求1-9中任一项所述的抗HER2抗体或其抗原结合片段、或权利要求10-18中任一项所述的抗HER2互补性双特异抗体与所述样品或分离自所述患者的生物样本进行孵育，检测所述抗体是否结合至所述样品或生物样本。
PCT/CN2024/097980 2023-06-09 2024-06-07 抗her2互补性双特异抗体-药物偶联物及其制备方法和用途 Ceased WO2024251240A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202480038349.6A CN121311508A (zh) 2023-06-09 2024-06-07 抗her2互补性双特异抗体-药物偶联物及其制备方法和用途
EP24818770.0A EP4725969A1 (en) 2023-06-09 2024-06-07 Anti-her2 complementary bispecific antibody-drug conjugate, preparation method therefor and use thereof

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN202310684362 2023-06-09
CN202310684362.6 2023-06-09
CN202311002749.5 2023-08-10
CN202311002749 2023-08-10
CN202311490098.9 2023-11-09
CN202311490098 2023-11-09
CN202410237969.4 2024-03-01
CN202410237969 2024-03-01

Publications (1)

Publication Number Publication Date
WO2024251240A1 true WO2024251240A1 (zh) 2024-12-12

Family

ID=93795050

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/097980 Ceased WO2024251240A1 (zh) 2023-06-09 2024-06-07 抗her2互补性双特异抗体-药物偶联物及其制备方法和用途

Country Status (4)

Country Link
EP (1) EP4725969A1 (zh)
CN (1) CN121311508A (zh)
TW (1) TW202513596A (zh)
WO (1) WO2024251240A1 (zh)

Citations (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1990005144A1 (en) 1988-11-11 1990-05-17 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
US5202238A (en) 1987-10-27 1993-04-13 Oncogen Production of chimeric antibodies by homologous recombination
US5204244A (en) 1987-10-27 1993-04-20 Oncogen Production of chimeric antibodies by homologous recombination
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5374548A (en) 1986-05-02 1994-12-20 Genentech, Inc. Methods and compositions for the attachment of proteins to liposomes using a glycophospholipid anchor
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
US5399331A (en) 1985-06-26 1995-03-21 The Liposome Company, Inc. Method for protein-liposome coupling
US5416016A (en) 1989-04-03 1995-05-16 Purdue Research Foundation Method for enhancing transmembrane transport of exogenous molecules
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5622699A (en) 1995-09-11 1997-04-22 La Jolla Cancer Research Foundation Method of identifying molecules that home to a selected organ in vivo
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO1999065894A1 (en) 1998-06-17 1999-12-23 Eisai Co., Ltd. Macrocyclic analogs and methods of their use and preparation
US6068829A (en) 1995-09-11 2000-05-30 The Burnham Institute Method of identifying molecules that home to a selected organ in vivo
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO2003106496A1 (es) 2002-06-14 2003-12-24 Consejo Superior De Investigaciones Científicas Anticuerpos, péptidos y vacunas activos frente al virus vih-1
WO2004034990A2 (en) 2002-10-16 2004-04-29 Eisai Co., Ltd. Methods and compositions for use in treating cancer
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
WO2011014469A1 (en) 2009-07-29 2011-02-03 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human angiopoietin-2
US8163888B2 (en) 2003-10-10 2012-04-24 Immunogen, Inc. Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates
US8624003B2 (en) 2009-06-03 2014-01-07 Immunogen, Inc. Methods for preparation of antibody-maytansinoid conjugates
WO2014060365A1 (en) * 2012-10-15 2014-04-24 Universität Zürich Prorektorat Mnw Bispecific her2 ligands for cancer therapy
WO2017151979A1 (en) 2016-03-02 2017-09-08 Eisai Inc. Eribulin-based antibody-drug conjugates and methods of use
US20190127481A1 (en) * 2012-10-15 2019-05-02 Universität Zürich Bispecific HER2 Ligands for Cancer Therapy
CN114805590A (zh) * 2021-01-22 2022-07-29 三生国健药业（上海）股份有限公司 结合her2的双特异性抗体
WO2023041006A1 (zh) * 2021-09-16 2023-03-23 正大天晴药业集团股份有限公司 抗her3抗体药物偶联物及其组合物和用途

Patent Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
US5399331A (en) 1985-06-26 1995-03-21 The Liposome Company, Inc. Method for protein-liposome coupling
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5374548A (en) 1986-05-02 1994-12-20 Genentech, Inc. Methods and compositions for the attachment of proteins to liposomes using a glycophospholipid anchor
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US5204244A (en) 1987-10-27 1993-04-20 Oncogen Production of chimeric antibodies by homologous recombination
US5202238A (en) 1987-10-27 1993-04-13 Oncogen Production of chimeric antibodies by homologous recombination
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1990005144A1 (en) 1988-11-11 1990-05-17 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US5416016A (en) 1989-04-03 1995-05-16 Purdue Research Foundation Method for enhancing transmembrane transport of exogenous molecules
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
US5399163A (en) 1992-07-24 1995-03-21 Bioject Inc. Needleless hypodermic injection methods and device
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5622699A (en) 1995-09-11 1997-04-22 La Jolla Cancer Research Foundation Method of identifying molecules that home to a selected organ in vivo
US6068829A (en) 1995-09-11 2000-05-30 The Burnham Institute Method of identifying molecules that home to a selected organ in vivo
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO1999065894A1 (en) 1998-06-17 1999-12-23 Eisai Co., Ltd. Macrocyclic analogs and methods of their use and preparation
WO2003106496A1 (es) 2002-06-14 2003-12-24 Consejo Superior De Investigaciones Científicas Anticuerpos, péptidos y vacunas activos frente al virus vih-1
WO2004034990A2 (en) 2002-10-16 2004-04-29 Eisai Co., Ltd. Methods and compositions for use in treating cancer
US8163888B2 (en) 2003-10-10 2012-04-24 Immunogen, Inc. Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
US8624003B2 (en) 2009-06-03 2014-01-07 Immunogen, Inc. Methods for preparation of antibody-maytansinoid conjugates
WO2011014469A1 (en) 2009-07-29 2011-02-03 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human angiopoietin-2
WO2014060365A1 (en) * 2012-10-15 2014-04-24 Universität Zürich Prorektorat Mnw Bispecific her2 ligands for cancer therapy
US20190127481A1 (en) * 2012-10-15 2019-05-02 Universität Zürich Bispecific HER2 Ligands for Cancer Therapy
WO2017151979A1 (en) 2016-03-02 2017-09-08 Eisai Inc. Eribulin-based antibody-drug conjugates and methods of use
CN114805590A (zh) * 2021-01-22 2022-07-29 三生国健药业（上海）股份有限公司 结合her2的双特异性抗体
WO2023041006A1 (zh) * 2021-09-16 2023-03-23 正大天晴药业集团股份有限公司 抗her3抗体药物偶联物及其组合物和用途

Non-Patent Citations (67)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NP_004439.2
"PCR Protocols: A Guide to Methods and Applications", 1990, ACADEMIC PRESS
"Remington: The Science and Practice of Pharmacy", 2003, LIPPINCOTT WILLIAMS & WILKINS
"Sustained and Controlled Release Drug Delivery Systems", 1978, MARCEL DEKKER
AL-LAZIKANI ET AL., J MOL BIOL, vol. 273, 1997, pages 927 - 748
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 3389 - 3402
ARAP ET AL., SCIENCE, vol. 279, 1998, pages 377 - 380
BLOEMAN ET AL., FEBS LETT, vol. 357, 1995, pages 140 - 144
BRISCOE ET AL., AM J PHYSIOL, vol. 268, 1995, pages L374 - 380
CHO ET AL., NATURE, vol. 421, 2003, pages 756 - 760
CHOTHIA ET AL., NATURE, vol. 341, 1989, pages 544 - 546
CHOTHIALESK, J MOL BIOL, vol. 196, 1987, pages 901 - 917
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
COX ET AL., EUR J IMMUNOL, vol. 24, 1994, pages 827 - 836
FITTING ET AL., MABS, vol. 7, 2015, pages 390 - 402
FRANKLIN ET AL., CANCER CELL, vol. 5, 2004, pages 317 - 328
GARRETT ET AL., MOL CELL, vol. 11, 2003, pages 495 - 505
GHETIE ET AL., ANNU REV IMMUNOL, vol. 18, 2000, pages 739 - 766
GRAHAM ET AL., J GEN VIROL, vol. 36, 1977, pages 59 - 74
HAMANN ET AL., EXPERT OPINION ON THERAPEUTIC PATENTS, vol. 15, 2005, pages 1087 - 1103
HAMBLETT, K.J. ET AL.: "Abstract P 6-17 -13: ZW 49, a HER2 Targeted Biparatopic Antibody Drug Conjugate for the Treatment of HER2 Expressing Cancers", CANCER RESEARCH, vol. 79, 15 February 2019 (2019-02-15), XP009542493, DOI: 10.1158/1538-7445.SABCS18-P6-17-13 *
HE ET AL., J NUCL MED, vol. 51, 2010, pages 427 - 432
HIGGINS ET AL., METHODS IN ENZYMOLOGY, vol. 266, 1996, pages 383 - 402
HOLLIGER ET AL., PROC NATL ACAD SCI USA, vol. 90, 1993, pages 6444 - 6448
HUSTON ET AL., PROC NATL ACAD SCI USA, vol. 85, 1988, pages 5879 - 5883
JAIN ET AL., PHARMACEUTICAL RESEARCH, vol. 32, 2015, pages 3526 - 3540
JEFFERIS ET AL., IMMUNOL LETT, vol. 82, 2002, pages 57 - 65
JOHNSON ET AL., NUCLEIC ACIDS RES, vol. 29, 2001, pages 207 - 209
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KABAT ET AL.: "NIH Publication No. 91-3242", 1991, article "Sequences of Proteins of Immunological Interest"
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", NIH PUBLICATION NO. 91-3242, 1991
KEINANENLAUKKANEN, FEBS LETT, vol. 346, 1994, pages 123 - 126
KETCHEMEN, J.P . ET AL.: "Biparatopic Anti-HER2 Drug Radioconjugates as Breast Cancer Theranostics", BRITISH JOURNAL OF CANCER, vol. 129, 24 April 2023 (2023-04-24), pages 153 - 162, XP093239753, DOI: 10.1038/s41416-023-02272-4 *
KILLIONFIDLER, IMMUNOMETHODS, vol. 4, 1994, pages 273 - 279
KLEIN ET AL., MABS, vol. 4, 2012, pages 653 - 663
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
LARKIN ET AL., BIOINFORMATICS, vol. 23, 2007, pages 2947 - 2948
LEFRANC MP ET AL., DEV COMP IMMUNOL, vol. 27, 2003, pages 55 - 77
LEFRANC MP ET AL., NUCLEIC ACIDS RESEARCH, vol. 27, 1999, pages 209 - 212
LEFRANC MP, THE IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
MACCALLUM RM ET AL., J MOL BIOL, vol. 262, 1996, pages 732 - 745
MARKS ET AL., J MOL BIOL, vol. 222, 1991, pages 581 - 597
MARTIN ET AL., METHODS ENZYMOL, vol. 203, 1991, pages 121 - 153
MARTIN ET AL., PROC NATL ACAD SCI USA, vol. 86, 1989, pages 10029 - 10033
MATHER ET AL., ANN NY ACAD SCI, vol. 383, 1982, pages 44 - 68
MATHER, BIOL REPROD, vol. 23, 1980, pages 243 - 251
MODI ET AL., N ENGL J MED, vol. 7, 8 March 2022 (2022-03-08), pages 9 - 20
MOHAN ET AL., MOLECULAR CANCER THERAPEUTICS, vol. 15, 2016, pages 1321 - 1331
MORRISON SL ET AL., PROC NATL ACAD SCI USA, vol. 81, 1984, pages 6851 - 6855
OGANESYAN ET AL., ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 64, 2008, pages 700 - 704
OWAIS ET AL., ANTIMICROB AGENTS CHEMOTHER, vol. 39, 1995, pages 180 - 184
PEGRAM, M.D ET AL.: "First-in-human, Phase 1 Dose-Escalation Study of Biparatopic Anti- HER2 Antibody-Drug Conjugate MEDI4276 in Patients with HER2-positive Advanced Breast or Gastric Cancer", MOLECULAR CANCER THERAPEUTICS, vol. 20, no. 8, 31 August 2021 (2021-08-31), pages 1442 - 1453, XP093023048, DOI: 10.1158/1535-7163.MCT-20-0014 *
POLJAK ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 1123
RAGHAVAN ET AL., ANNU REV CELL DEV BIOL, vol. 12, 1996, pages 181 - 220
RANADE, J CLIN PHARMACOL, vol. 29, 1989, pages 685 - 694
RAVETCH ET AL., ANNU REV IMMUNOL, vol. 19, 2001, pages 275 - 290
RIECHMANN ET AL., NATURE, vol. 332, 1998, pages 323 - 327
RUIZ ET AL., NUCLEIC ACIDS RES, vol. 28, 2000, pages 219 - 221
SAIKI ET AL., SCIENCE, vol. 239, 1988, pages 487 - 491
SCHREIER ET AL., J BIOL CHEM, vol. 269, 1994, pages 9090 - 9098
SMITH ET AL., SCIENCE, vol. 228, 1985, pages 1315 - 1317
TOMLINSON ET AL., J MOL BIOL, vol. 227, 1992, pages 776 - 798
UMEZAWA ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 153, 1988, pages 1038 - 1044
URLAUB ET AL., PROC NATL ACAD SCI USA, vol. 77, 1980, pages 4216 - 4220
WARD ET AL., THER IMMUNOL, vol. 2, 1995, pages 77 - 94
ZAPATA ET AL., PROTEIN ENG, vol. 8, 1995, pages 1057 - 1062
ZHOU ET AL., MOL CANCER THER, vol. 11, 2012, pages 1167 - 1476

Also Published As

Publication number Publication date
TW202513596A (zh) 2025-04-01
CN121311508A (zh) 2026-01-09
EP4725969A1 (en) 2026-04-15

Similar Documents

Publication Publication Date Title
CN114502592B (zh) 靶向cldn18.2的抗体及其制备方法和应用
US20230272063A1 (en) Anti-claudin 18 antibodies and methods of use thereof
US9689862B2 (en) Antigen binding protein and its use as addressing product for the treatment of cancer
KR20230062600A (ko) 항-ceacam5 항체 및 접합체 및 이의 용도
AU2021334677A1 (en) Anti-ROR1 antibodies and related bispecific binding proteins
JP2021528973A (ja) 抗ｓｔｅａｐ１抗原結合タンパク質
JP2025508009A (ja) ＦＲαに対する結合分子
US12428479B2 (en) CLDN18.2-targeting antibody, bispecific antibody and use thereof
US12509513B2 (en) Anti-L1CAM antibodies and uses thereof
WO2022143670A1 (zh) 结合trop2的抗体及其用途
US20250236682A1 (en) Antigen-binding protein comprising two fc domains and use thereof
KR20230154020A (ko) 클라우딘-6에 대한 항체 및 이의 용도
TW202227498A (zh) 新型抗ｃｌａｕｄｉｎ１８抗體
JP2026065117A (ja) 最適化された薬物コンジュゲーションのための改変された結合ポリペプチド
TW202513596A (zh) 抗ｈｅｒ２抗體、抗體－藥物偶聯物及其藥物組合物、藥盒、製備方法和用途
WO2025237392A1 (zh) 新型抗egfr抗体、药物偶联物及其用途
WO2026001924A1 (zh) 结合gprc5d的抗体及其用途
TW202430568A (zh) 抗b7h3抗體以及其使用方法
CN120271715A (zh) 抗体及其药物偶联物和用途
HK40120932A (zh) 抗体及其药物偶联物和用途
HK40105064A (zh) Folr1结合剂、其偶联物及其使用方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24818770

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2025571536

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2025571536

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2024818770

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024818770

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024818770

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024818770

Country of ref document: EP

Effective date: 20260109

WWP Wipo information: published in national office

Ref document number: 2024818770

Country of ref document: EP