WO2022127889A1 - Her2抗体及其应用 - Google Patents

Her2抗体及其应用 Download PDF

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WO2022127889A1
WO2022127889A1 PCT/CN2021/139018 CN2021139018W WO2022127889A1 WO 2022127889 A1 WO2022127889 A1 WO 2022127889A1 CN 2021139018 W CN2021139018 W CN 2021139018W WO 2022127889 A1 WO2022127889 A1 WO 2022127889A1
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seq
antibody
antigen
numbering system
cancer
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PCT/CN2021/139018
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French (fr)
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邵小慧
杨翠青
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to US18/267,561 priority Critical patent/US20240076403A1/en
Priority to CN202180083703.3A priority patent/CN116829587A/zh
Publication of WO2022127889A1 publication Critical patent/WO2022127889A1/zh

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], 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
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present disclosure relates to the field of antibodies, in particular to HER2 antibodies and applications thereof.
  • HER2 is a receptor tyrosine kinase located on the cell membrane surface encoded by the HER2/neu gene, belonging to the epidermal growth factor receptor (EGFR) family, which is usually involved in the signaling of cell growth and differentiation.
  • HER2 has no natural ligand, but can be activated by overexpression of homodimers or by heterodimerization with other members of the HER family that can be activated by ligand binding, thereby activating receptor tyrosine kinases, Through mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase-protein kinase B/Akt (PI3K-PKB/Akt), phospholipase C-protein kinase C (PLC-PKC) and transcriptional signal transduction and Activated protein (STAT) and other signaling pathways trigger downstream signaling cascades (Olayioye MA, Breast Cancer Res.
  • EGFR epidermal growth factor receptor
  • HER2 is expressed at low levels in very few tissues and is overexpressed in more than 30% of human tumors.
  • the overexpression of HER2 gene is not only related to the occurrence and development of tumors, but also an important clinical treatment detection and prognostic indicator, and an important target for tumor targeted therapy drug selection (Cho HS and Leahy DJ, Science. 2002; 297( 5585):1330-1333).
  • Trastuzumab (trade name, Herceptin, Herceptin) is a recombinant humanized monoclonal anti-HER2 antibody approved by the US FDA in 1998. It blocks ligand-independent HER2 homodimerization and, to a lesser extent, heterodimerization with other HER family members (Cho et al., Nature. 2003; 421:756-760; Wehrman et al., Proc. Natl Acad Sci USA. 2006;103(50):19063-19068).
  • trastuzumab which is highly effective in patients with HER2-overexpressing primary invasive breast cancer, is effective in breast cancer tumors with high HER2 expression, but is limited by high HER2 expression and the potential for relapse in initial responders (Dinh et al., Clin Adv Hematol Oncol. 2007;5(9):707-717).
  • Pertuzumab (trade name, Perjeta) is another humanized monoclonal anti-HER2 antibody. It targets domain II of the HER2 protein that dimerizes, thereby blocking the formation of HER2 heterodimers (Hughes et al., Mol Cancer Ther. 2009;8(7):1885-1892). Pertuzumab, which loosely requires high levels of HER2 expression, offers more treatment options for patients with HER2-low breast cancer (Franklin et al., Cancer Cell. 2004;5(4):317-328). The combination of Pertuzumab and Trastuzumab can enhance the antitumor effect (Baselga et al., J Clin Oncol. 2010;28:1138-1144).
  • HCAbs heavy chain antibodies
  • This type of antibody contains only one heavy chain variable region and two conventional CH2 and CH3 regions, but it has good Structural stability and antigen-binding activity.
  • Heavy chain antibodies and the VHH domains derived therefrom have the advantages of small molecular weight, flexible chemical properties, easy expression, good solubility, strong permeability, weak immunogenicity, simple humanization, and easy coupling to other molecules, making up for the traditional Antibody deficiencies also increase the diversity of drug development. Therefore, there is an urgent need in the art to develop new and effective specific VHH domain or heavy chain antibodies against HER2.
  • the present disclosure provides an antibody or antigen-binding fragment that specifically binds to HER2, a multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid fragment, a vector, a host cell, a pharmaceutical composition, a kit, and a preparation method and its application in the treatment of diseases and detection of HER2.
  • the present invention relates to an antibody or antigen-binding fragment that specifically binds HER2, said antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system; alternatively, the HCDR1, HCDR2 and HCDR3 are selected from Table 9;
  • the HCDR1 is selected from the group consisting of SEQ ID NOs: 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82 , 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157 , 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190 or 193;
  • the HCDR2 is selected from SEQ ID NOs: 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83 , 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158 , 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191 or 194;
  • the HCDR3 is selected from the group consisting of SEQ ID NOs: 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 , 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159 , 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192 or 195.
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 34-36, SEQ ID NO: 37-39 or SEQ ID NO: 40-42;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 43-45, SEQ ID NO: 46-48 or SEQ ID NO: 49-51;
  • the Kabat numbering system or the Chothia numbering system are selected from SEQ ID NO: 52-54, SEQ ID NO: 55-57 or SEQ ID NO: 58-60;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 61-63, SEQ ID NO: 64-66 or SEQ ID NO: 67-69;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 70-72, SEQ ID NO: 73-75 or SEQ ID NO: 76-78;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 79-81, SEQ ID NO: 82-84 or SEQ ID NO: 85-87;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 88-90, SEQ ID NO: 91-93 or SEQ ID NO: 94-96;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 97-99, SEQ ID NO: 100-102 or SEQ ID NO: 103-105;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 106-108, SEQ ID NO: 109-111 or SEQ ID NO: 112-114;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 115-117, SEQ ID NO: 118-120 or SEQ ID NO: 121-123;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 124-126, SEQ ID NO: 127-129 or SEQ ID NO: 130-132;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 133-135, SEQ ID NO: 136-138 or SEQ ID NO: 139-141;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 142-144, SEQ ID NO: 145-147 or SEQ ID NO: 148-150;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 151-153, SEQ ID NO: 154-156 or SEQ ID NO: 157-159;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 160-162, SEQ ID NO: 163-165 or SEQ ID NO: 166-168;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 169-171, SEQ ID NO: 172-174 or SEQ ID NO: 175-177;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 178-180, SEQ ID NO: 181-183 or SEQ ID NO: 184-186;
  • the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 187-189, SEQ ID NO: 190-192 or SEQ ID NO: 193-195 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.
  • said CDR1, CDR2 and/or CDR3 comprises at most 10, 9, 8, 7, 6, 5, 4 occurrences on said HCDR1, HCDR2 and/or HCDR3 1, 3, 2 or 1 mutated amino acid sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably the substitutions are conservative amino acid substitutions.
  • said CDR1, CDR2 and/or CDR3 comprise at least 80%, 85%, 90%, 91%, 92%, 93%, Sequences of 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment comprises a single domain antibody comprising the CDRl, CDR2 and CDR3.
  • the single-domain antibody comprises a sequence selected from any one of SEQ ID NOs: 16-33; optionally, the single-domain antibody comprises any of SEQ ID NOs: 16-33 Sequences indicated by an item occur up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 A sequence of 1, 6, 5, 4, 3, 2 or 1 mutations selected from insertions, deletions and/or substitutions, preferably conservative amino acid substitutions; optionally, The single domain antibody comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% compared with the sequence shown in any one of SEQ ID NOs: 16 to 33 Sequences of %, 97%, 98%, 99% or 100% identity.
  • the antibody comprises the FR region in the VHH domain shown in any one of SEQ ID NOs: 16-33;
  • the FR regions in the VHH domains shown in item occur at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated sequence, the mutation is selected from insertion, deletion and/or replacement, and the replacement is preferably a conservative amino acid replacement; optionally, the antibody comprises and SEQ ID NO: 16 ⁇ 33
  • the FR regions in the VHH domains shown in any one of them have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Sequences that are 99% or 100% identical.
  • the antibody or antigen-binding fragment is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.
  • the antibody or antigen-binding fragment contains or does not contain an antibody heavy chain constant region; alternatively, the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat, Rabbit or sheep; alternatively, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain The constant region can be selected from an Fc region, a CH3 region or an intact heavy chain constant region, preferably, the heavy chain constant region is a human Fc region; preferably, the antibody or antigen-binding fragment is a heavy chain antibody.
  • the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat, Rabbit or sheep; alternatively, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be selected from IgG1, IgG2,
  • the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents
  • the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the antibody or antigen-binding fragment specifically binds human HER2, monkey HER2 and/or murine HER2, preferably, the antibody or antigen-binding fragment binds to the KD of human HER2, monkey HER2 and murine HER2 Less than 1E-6M, 1E-7M, 2E-7M, 3E-7M, 4E-7M, 5E-7M, 6E-7M, 8E-7M, 9E-7M, 1E-8M, 2E-8M, 3E-8M, 4E -8M, 5E-8M, 6E-8M, 8E-8M, 9E-8M, ;1E-9M, 2E-9M, 3E-9M, 4E-9M, 5E-9M, 6E-9M, 8E-9M, 9E- 9M, 1E-10M or 1E-11M.
  • the present disclosure relates to an antibody or antigen-binding fragment that specifically binds Her2, said antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 respectively comprising selected from SEQ ID NO: HCDR1, HCDR2, and HCDR3 of the VHH domains set forth in any one of 16 to 33.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system; alternatively, the HCDR1, HCDR2 and HCDR3 are selected from Table 9;
  • the HCDR1 is selected from SEQ ID NOs: 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88 , 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163 , 166, 169, 172, 175, 178, 181, 184, 187, 190 or 193;
  • the HCDR2 is selected from SEQ ID NOs: 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89 , 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164 , 167, 170, 173, 176, 179, 182, 185, 188, 191 or 194;
  • the HCDR3 is selected from SEQ ID NOs: 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90 , 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165 , 168, 171, 174, 177, 180, 183, 186, 189, 192 or 195;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 34-36, SEQ ID NO: 37-39 or SEQ ID NO: 40-42;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 43-45, SEQ ID NO: 46-48 or SEQ ID NO: 49-51;
  • the Kabat numbering system or the Chothia numbering system are selected from SEQ ID NO: 52-54, SEQ ID NO: 55-57 or SEQ ID NO: 58-60;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 61-63, SEQ ID NO: 64-66 or SEQ ID NO: 67-69;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 70-72, SEQ ID NO: 73-75 or SEQ ID NO: 76-78;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 79-81, SEQ ID NO: 82-84 or SEQ ID NO: 85-87;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 88-90, SEQ ID NO: 91-93 or SEQ ID NO: 94-96;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 97-99, SEQ ID NO: 100-102 or SEQ ID NO: 103-105;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 106-108, SEQ ID NO: 109-111 or SEQ ID NO: 112-114;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 115-117, SEQ ID NO: 118-120 or SEQ ID NO: 121-123;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 124-126, SEQ ID NO: 127-129 or SEQ ID NO: 130-132;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 133-135, SEQ ID NO: 136-138 or SEQ ID NO: 139-141;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 142-144, SEQ ID NO: 145-147 or SEQ ID NO: 148-150;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 151-153, SEQ ID NO: 154-156 or SEQ ID NO: 157-159;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 160-162, SEQ ID NO: 163-165 or SEQ ID NO: 166-168;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 169-171, SEQ ID NO: 172-174 or SEQ ID NO: 175-177;
  • the Kabat numbering system or the Chothia numbering system the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 178-180, SEQ ID NO: 181-183 or SEQ ID NO: 184-186;
  • the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 187-189, SEQ ID NO: 190-192 or SEQ ID NO: 193-195 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.
  • said CDR1, CDR2 and/or CDR3 comprises at most 10, 9, 8, 7, 6, 5, 4 occurrences on said HCDR1, HCDR2 and/or HCDR3 1, 3, 2 or 1 mutated amino acid sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably the substitutions are conservative amino acid substitutions.
  • said CDR1, CDR2 and/or CDR3 comprises at least 80, 85%, 90%, 91%, 92%, 93%, 94% compared to said HCDR1, HCDR2 and/or HCDR3 Sequences of %, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment competes with trastuzumab, pertuzumab, or FRP5 mAb for binding to HER2, or the antibody or antigen-binding fragment does not bind to trastuzumab Anti-, Pertuzumab or FRP5 mAbs compete for binding to HER2.
  • the antibody or antigen-binding fragment is further linked with other functional molecules, preferably, the other functional molecules can be selected from one or more of the following: signal peptide, protein tag, cell factors, angiogenesis inhibitors, or immune checkpoint inhibitors.
  • the cytokine can be IL2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha;
  • the angiogenesis inhibitor can be endostatin;
  • the immune checkpoint inhibitor can be SIRP ⁇ .
  • the present disclosure also discloses a multispecific antigen-binding molecule, the multispecific antigen-binding molecule comprising the aforementioned antibody or antigen-binding fragment, and an antigen-binding molecule that binds to other antigens other than HER2, or that binds to the aforementioned antibody or antigen-binding fragment.
  • HER2 epitopes of antibodies or antigen-binding fragments can be selected from: CD3, preferably CD3 ⁇ ; CD16, preferably CD16A; CD137; CD258; 4-1BB; CD40; CD64; EGFR; HER1; HER3; PD-1; PD-L1; VEGF; IGF-IR (insulin-like growth factor type I receptor); Phosphatidylserine (PS); c-Met or blood-brain barrier receptor;
  • the other antigen-binding molecules are antibodies or antigen-binding fragments
  • the multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific;
  • the multispecific antigen binding molecule may be bivalent, tetravalent or hexavalent.
  • the present disclosure also discloses a chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding
  • the domains comprise the aforementioned antibodies or antigen-binding fragments.
  • the present disclosure also discloses an immune effector cell, the immune effector cell expressing the aforementioned chimeric antigen receptor, or comprising a nucleic acid fragment encoding the aforementioned chimeric antigen receptor; preferably, the immune effector cell selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, the T cells
  • the cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present disclosure also discloses an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule or chimeric antigen receptor.
  • the present disclosure also discloses a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also discloses a host cell, the host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insects cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insects cells or mammalian cells (CHO cell line or 293T cell line).
  • the present disclosure also discloses a method for preparing the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell specific antigen binding molecules.
  • the present disclosure further discloses a method for preparing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, optionally, the method further comprising activating the immune effector cells
  • the effector cells express the aforementioned CAR.
  • the present disclosure also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, carrier or prepared according to the aforementioned method
  • the obtained product optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present disclosure also discloses a method of treating a tumor or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, immune effector cell, Nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods; preferably, the tumor or cancer is selected from solid tumors, gastric cancer, gastroesophageal junction cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, Prostate cancer, castration-resistant prostate cancer, breast cancer, HER2-positive breast cancer, sarcoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, bladder cancer, esophageal cancer, colon cancer Rectal cancer, head and neck cancer, salivary gland cancer, or B-cell acute lymphoblastic leukemia.
  • the tumor or cancer is selected from solid tumors, gas
  • the present disclosure also discloses that the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, immune effector cells, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods are used in the preparation of the treatment of tumors or cancers Use in medicine; preferably, the tumor or cancer is selected from solid tumors, gastric cancer, gastroesophageal junction cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, prostate cancer, castration-resistant prostate cancer, breast cancer, HER2-positive breast cancer, sarcoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, bile duct cancer, urothelial cancer, bladder cancer, esophageal cancer, colorectal cancer, head and neck cancer, salivary gland cancer, or B cell cancer acute lymphoblastic leukemia.
  • the tumor or cancer is selected from solid tumors, gastric cancer, gastroesophageal junction
  • the present disclosure also discloses the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, immune effector cells, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods, for treating tumors or cancer; preferably, the tumor or cancer is selected from solid tumors, gastric cancer, gastroesophageal junction cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, prostate cancer, castration-resistant prostate cancer, breast cancer, HER2 positive Breast cancer, sarcoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, bladder cancer, esophageal cancer, colorectal cancer, head and neck cancer, salivary gland cancer, or B cell acute lymphoma cell leukemia.
  • the tumor or cancer is selected from solid tumors, gastric cancer, gastroesophageal junction cancer, ovarian cancer, fallopia
  • the present disclosure also discloses a kit comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, carrier, and the aforementioned antibody or antigen-binding fragment prepared according to the aforementioned method. product or pharmaceutical composition.
  • the present disclosure further discloses a method for detecting the expression of HER2 in a biological sample, the method comprising causing the biological sample to form a complex between the aforementioned antibody or antigen-binding fragment and HER2, allowing the biological sample to form a complex.
  • the present disclosure also discloses the use of the aforementioned antibody or antigen-binding fragment in the preparation of a HER2 detection reagent.
  • Term "Her2" herein also known as ErbB2, NEU or CD340, which includes human epidermal growth factor receptor 2 (UniProtKB ID: P04626-1) and its mutants, isoforms and species Sources (species homologs).
  • Species homologues of HER2 herein include, but are not limited to, vertebrate or mammalian HER2 and mutants and isoforms thereof, eg, monkey Her2 (NCBI ID: XP_014975023.1) and mouse Her2.
  • the term "specifically binds" herein refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • high affinity generally refers to having about 10-6 M or less, 10-7 M or less, about 10-8 M or less, about 1 ⁇ 10-9 M or less, about 1 ⁇ KD of 10-10 M or lower, 1 ⁇ 10-11 M or lower, or 1 ⁇ 10-12 M or lower.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the association rate.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
  • antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is The N-terminal to C-terminal direction consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, the hinge region (HR), the heavy chain constant region CH2 domain, the heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction
  • the polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure.
  • immunoglobulins Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ and ⁇ chains, respectively. , ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other camelids produced heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin receptor (Ig new antigen receptor, IgNAR).
  • HCAbs heavy-chain antibodies
  • heavy chain antibody herein refers to an antibody that lacks the light chain of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising the VH antigen binding domain and the CH2 and CH3 constant domains in the absence of the CH1 domain.
  • VHH domain and “nanobody” and “single domain antibody” (sdAb) herein have the same meaning and can be used interchangeably, and refer to the variable region of cloning heavy chain antibodies, constructing A single-domain antibody consisting of only one heavy chain variable region, it is the smallest fully functional antigen-binding fragment.
  • a heavy chain antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • an “antibody” herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • multispecific herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
  • the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
  • full-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean having a structure that is substantially similar to that of a native antibody.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein and do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody that have the ability to bind antigenic capacity.
  • antigen-binding fragments or “antibody fragments” herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domains Antibody.
  • chimeric antibody herein refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat, mouse, rabbit, or alpaca) and derived from a different organism (eg, human ) of the constant regions of immunoglobulins.
  • Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, Bio Techniques 4: 214-221; Gillies et al, 1985 J Immunol Methods 125: 191-202; into this article.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune responses, and the like.
  • Fully human antibody refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
  • “Heavy chain variable region” is used interchangeably with “VH” and “HCVR”.
  • VL is used interchangeably with "VL”, “LCVR”.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • the light chain variable region CDR can be abbreviated as LCDR.
  • framework region or "FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody.
  • CDRs may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues differently defined.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • IMGT International ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
  • the variable domains have more conserved amino acid sequences.
  • the “heavy chain constant region” may be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage, cleavage of one or more, particularly one or two amino acids, from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region comprises the IgG CH2 and IgG CH3 domains, optionally, the entire or partial hinge region, but not the CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • a CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain.
  • the "CH3 domain" comprises that stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • a CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole", hole) of the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • a variant CH3 domain eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole”, hole) of the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National As described in Institutes of Health, Bethesda, MD, 1991.
  • amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • identity herein can be calculated by aligning the sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (eg, it may be optimal). The alignment may introduce gaps in either or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences varies with the identical positions shared by the sequences.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com) using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences.
  • a particularly preferred set of parameters is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described in this disclosure can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • Such searches can be performed, for example, using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The heavy chain variable region and/or light chain variable region of (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is generally represented as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg, cDNA
  • RNA eg, mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP2101823B1).
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • pharmaceutical composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.
  • treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) undesired physiological changes or pathologies, such as cancers and tumors, in a subject being treated.
  • Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • subject herein refers to an organism receiving treatment for a particular disease or disorder as described herein.
  • a “subject” includes a mammal, such as a human, primate (eg, monkey) or non-primate mammal, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
  • Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • Fig. 1A shows the binding reaction of ELISA detection control antibody and human HER2-His protein
  • Fig. 1B shows the binding reaction of ELISA detection control antibody and monkey HER2-His protein
  • Figures 2A-2D are the FACS results of the control antibody detecting the expression of HER2 in SK-BR-3 cells;
  • Figures 3A-3F are the results of FACS screening of CHO-K1 cells transfected with human HER2 protein
  • Figure 4 is the FACS results of the Tab048 antibody detecting the expression of HER2 in HEK293T-monkey HER2 cells;
  • Figure 5A shows the serum antibody titer of alpaca after human HER2 protein immunization detected by ELISA
  • Figure 5B shows the serum antibody titer of alpaca after human HER2 protein immunization detected by FACS
  • 6A-6C are ELISA detection of the binding reaction of VHH-hFc and human HER2-His protein
  • Figures 7A-7C show the binding reaction of VHH-hFc to human HER2-CHO-K1 cells detected by ELISA
  • Figures 8A-8C show the binding reaction of VHH-hFc to SK-BR-3 tumor cells detected by ELISA
  • Figures 9A-9C show the binding reaction of VHH-hFc and monkey HER2-His protein detected by ELISA
  • Figures 10A-10C are ELISA detection of the binding reaction of VHH-hFc and murine HER2-His protein
  • Figures 11A-11C show the binding reaction of VHH-hFc to HEK293T-monkey HER2 cells detected by ELISA
  • Figures 12A-12B are SPR detection of the affinity of VHH-hFc and human HER2;
  • Figures 13A-13B are SPR detection of the affinity of VHH-hFc and monkey HER2;
  • Figure 14 shows the affinity of VHH-hFc and murine HER2 detected by SPR
  • Figure 15 is a competitive ELISA method to detect the inhibition rate between VHH-hFc;
  • VH and VL sequences of monoclonal antibodies FRP5, 4D5 (trastuzumab) and 2C4 (Pertuzumab) recognizing human HER2 were recombined into human IgG1 CH and CL expression vectors (wherein the expression vectors were purchased from Biointron, recombination step Also completed by Biointron), the recombinant plasmid was obtained (for the source of the experimental principle and steps of the above-mentioned plasmid recombination, see "Molecular Cloning Experiment Guide (Third Edition)", (US) J. Sambrook et al.).
  • VH and VL of cloned FRP5 were connected through three GGGGS linkers and recombined into the expression vector of human IgG1 Fc to obtain a recombinant plasmid, which was verified by sequencing.
  • Recombinant plasmids with a mass of 500 ⁇ g or more were extracted with an alkaline lysis method kit (purchased from QIAGEN) and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • the above plasmid construction and antibody expression and purification work were completed by Taizhou Baiying Biotechnology Co., Ltd.
  • 293F cells (purchased from Invitrogen) were cultured in medium 293medium (Medium A, purchased from Biointron). The shaker was set to 37°C, 120RPM and 8% CO2 (v/v). Mix 2 mL of medium A and 120 ⁇ l of 1 ⁇ g/mL PEI (purchased from Biointron) to obtain medium B. Mix 2 mL of medium A and 30 ⁇ g of recombinant plasmid to obtain medium C. After 5 minutes, the medium B and the medium C were combined and mixed, and allowed to stand for 15 minutes to obtain the mixed solution D.
  • the antibody of FRP5 human IgG1 format, the antibody of 4D5 human IgG1 format, the antibody of 2C4 human IgG1 format and the antibody of FRP5 ScFv-human IgG1 Fc (hFc) format are named Tab048, Tab049, Tab050 and Tab094 respectively, and the specific sequence information is shown in the table. 1 shown.
  • the binding activity of the control antibody to human HER2-His protein (purchased from Acro, product number: HE2-H5225) and monkey HER2-His protein (purchased from Sino Biological, product number: 90295-C08H) was detected by ELISA.
  • the specific detection results are shown in the table 2-3 and Figures 1A-1B, in which the IgG subtype control is hIgG1, and the graph data is the OD 450 value.
  • the results showed that Tab048, Tab049, Tab050, and Tab094 antibodies had good binding activity to human HER2 protein and monkey HER2 protein.
  • the SK-BR-3 cells were expanded and cultured in a T-75 cell culture flask to the logarithmic growth phase, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • Tab048, Tab049, Tab050 and Tab094 antibodies were used as primary antibodies, and APC-labeled secondary antibodies (purchased from Biolegend, product number: 409306) were detected and analyzed by FACS (FACS CantoTM, purchased from BD Company). The results are shown in Table 4 and Figures 2A-2D, indicating that SK-BR-3 cells can bind to Tab048, Tab049, Tab050 and Tab094.
  • the nucleotide sequence encoding the full-length amino acid sequence of human HER2 was cloned into pcDNA3.1 vector (purchased from Clontech) and prepare plasmids.
  • the CHO-K1 cell line purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences
  • was transfected with plasmids ( 3000 Transfection Kit, purchased from Invitrogen, Cat. No.
  • the nucleotide sequence encoding the full-length amino acid sequence of monkey HER2 (NCBI ID: XP_014975023.1, available at https://www.ncbi.nlm.nih.gov/protein/XP_014975023.1/) was cloned into the pcDNA3.1 vector ( was purchased from Thermofisher scientific) and plasmids were prepared.
  • HEK293T cell line HD Promega, Cat.
  • HER2 Thr 23-Thr 652-His protein (purchased from Acro, catalog number: HE2-H82E2).
  • human HER2-His protein was emulsified with Freund's complete adjuvant and then injected subcutaneously at multiple points, that is, 500 ⁇ g of human HER2-His protein was injected into each alpaca.
  • human HER2-His protein was emulsified with incomplete Freund's adjuvant and then injected subcutaneously at multiple points, that is, 250 ⁇ g of human HER2-His protein was injected into each alpaca.
  • RNAiso Plus reagent The extracted RNA was reverse transcribed into cDNA using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (purchased from Takara, Cat. No. 6210A). Amplification of nucleic acid fragments encoding heavy chain antibody variable regions by nested PCR:
  • Upstream primer CTTGGTGGTCCTGGCTGC (SEQ ID NO: 11);
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 12).
  • Upstream primer CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO: 13);
  • Downstream Primer-1 CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 14);
  • Downstream Primer-2 CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG (SEQ ID NO: 15).
  • the target single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pcomb3XSS using the restriction enzyme SfiI.
  • the product was then electrotransformed into E. coli electrocompetent cells TG1, and a single-domain antibody phage display library against HER2 was constructed and assayed.
  • the size of the library volume was calculated to be 3.4 ⁇ 10 9 .
  • 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
  • the human HER2-His protein was diluted with carbonate buffer with pH value of 9.6 to a final concentration of 5 ⁇ g/mL, and 100 ⁇ L/well was added to the enzyme-labeled wells.
  • Each protein was coated with 8 wells and coated overnight at 4°C; Discard the coating solution, wash 3 times with PBS, add 300 ⁇ L of 3% BSA-PBS blocking solution to each well, block at 37°C for 1 hour; wash 3 times with PBS, add 100 ⁇ L of phage library, and incubate at 37°C for 1 hour; aspirate unbound phage, Wash 6 times with PBST and 2 times with PBS; add 100 ⁇ L Gly-HCl eluate, incubate at 37°C for 8 minutes to elute the specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube, quickly Neutralize with 10 ⁇ L Tris-HCl neutralization buffer; take 10 ⁇ L for gradient dilution, measure
  • Dilute human HER2 protein and monkey HER2 protein with carbonate buffer with pH value of 9.6 to final concentrations of 2 ⁇ g/mL and 1 ⁇ g/mL add 100 ⁇ L wells to enzyme-labeled wells, and coat overnight at 4°C; discard the coating PBST, washed 3 times with PBST, added 300 ⁇ L of 5% skim milk to each well, blocked at 37°C for 1 hour; washed 3 times with PBST, added 50 ⁇ L of phage culture supernatant and 50 ⁇ L of 5% skim milk to each well, incubated at 37°C for 1 hour; Wash 5 times with PBST, add horseradish peroxidase-labeled anti-M13 antibody (diluted at 1:10000 with PBS), 100 ⁇ L/well, 37°C for 1 hour; wash the plate 6 times with PBST.
  • VHH-hFc The sequencing results were analyzed, an evolutionary tree was constructed according to the VHH-encoded protein sequence, and the sequences that were closer in the evolutionary tree were eliminated according to sequence similarity.
  • the following VHH antibodies were selected (see Table 8 for details), and their CDR regions were analyzed by bioinformatics methods , the specific results are shown in Table 9, and the subsequent production and identification of VHH-hFc was carried out.
  • the analysis methods include: Chothia definition and Kabat definition, using the following analysis websites: http://cao.labshare.cn/AbRSA/abrsa.php, http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi ; IMGT definition, using the following analysis website: http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results).
  • the target VHH sequence was recombined into the expression vector of human IgG1 Fc to obtain a recombinant plasmid.
  • the specific plasmid construction, transfection and purification procedures refer to Example 1(A).
  • VHH-hFc The purified VHH-hFc was analyzed for protein concentration, purity, and endotoxin (Lonza kit). The results are shown in Table 10. It was found that the final product of the antibody had a high purity, and the endotoxin concentration was within 1.0EU/mg.
  • Human HER2 protein was diluted with PBS to a final concentration of 2 ⁇ g/mL and then added to a 96-well ELISA plate at 50 ⁇ l per well. Cover with plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/w) BSA] and block for 2 hours at room temperature. The blocking solution was discarded and 50 ⁇ l of 100 nM serially diluted VHH-hFc or control antibody was added to each well. After incubation at 37°C for 2 hours, the plate was washed 3 times with PBS.
  • HRP horseradish peroxidase-labeled secondary antibody (purchased from Sigma, catalog number: A0170) was added, and after incubation at 37°C for 1 hour, the plate was washed 5 times with PBS. 50 ⁇ l of TMB substrate was added to each well, and after 10 minutes of incubation at room temperature, 50 ⁇ l of stop solution (1.0 M HCl) was added to each well. The OD 450nm value was read with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results of the binding activity of VHH-Fc to human HER2 protein are shown in Figures 6A-6C and Table 11, indicating that the purified antibody Binds to human HER2 protein.
  • the IgG control is hIgG1
  • the data in the table is the OD 450nm value
  • N/A means there is no data here.
  • Table 11 ELISA detects the binding reaction of VHH-hFc and human HER2 protein
  • the desired cells were expanded to logarithmic growth phase in T-75 cell culture flasks, the medium was aspirated, washed twice with PBS buffer, the cells were trypsinized, then the digestion was terminated with complete medium, and the cells were pipetted to single-cell suspension. After cell counting, centrifuge, resuspend the cell pellet with FACS buffer (PBS+2% fetal bovine serum) to 2x10 6 cells per ml, add 50 ⁇ l per well to 96-well FACS reaction plate, add VHH-hFc or control Antibodies were incubated at 50 ⁇ l per well for 1 hour at 4°C.
  • FACS buffer PBS+2% fetal bovine serum
  • the cells were centrifuged and washed three times with PBS buffer, 50 ⁇ l of FITC-labeled secondary antibody (purchased from Invitrogen, catalog number: A18830) was added to each well, and incubated on ice for 1 hour.
  • the cells were centrifuged and washed 3 times with PBS buffer, and 100 ⁇ l was detected and analyzed by FACS (FACS CantoTM, purchased from BD Company). Data analysis was performed by software (FlowJo) to obtain the mean fluorescence intensity (MFI) of the cells. Then, it was analyzed by software (GraphPad Prism8), data fitting was performed, and EC50 was calculated.
  • Monkey HER2-His protein purchased from Sino Biological, product number: 90295-C08H
  • murine HER2-his protein purchased from Sino Biological, product number: 50714-M08H
  • the analysis results are shown in Figures 9A-9C, Figures 10A-10C and Tables 13-14, all VHH-hFc can bind to monkey HER2 protein, and 7 VHH-hFc can bind to murine HER2 protein.
  • the IgG control is hIgG1
  • the data in the table is the OD 450nm value
  • N/A means there is no data here.
  • the HEK293T-monkey HER2 cells were subjected to FACS detection and data analysis according to the method of Example 4(B). The analysis results are shown in Table 15 and Figures 11A-11C, all VHH-hFcs had binding activity to HEK293T-monkey-HER2 cells.
  • Anti-human HER2 VHH-hFc antibodies were captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25 °C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, then a single concentration of HER2 antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration. Binding was measured by injecting different concentrations of human HER2-His in solution for 240 sec with a flow rate of 30 ⁇ L/min, starting at 200 nM (see detailed results for actual concentrations tested), diluted 1:1 for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer.
  • VHH-hFc and monkey HER2-His protein were carried out according to the method of Example 6(A), wherein antibodies Tab048 and Tab094 were used as controls. As shown in Table 17 and Figures 13A-13B, except for NB146-27, NB146-62, NB147-137 and NB147-161, VHH-hFc had better affinity with monkey HER2 protein.
  • VHH-hFc and murine HER2-His protein were carried out according to the method of Example 6(A), in which the antibody Tab094 was used as a control. As shown in Table 18 and Figure 14, NB147-32, NB147-36, NB147-66 and NB147-61 have good affinity with murine HER2 protein.
  • the HER2 VHH-hFc was grouped by a competition ELISA method. According to the method of Example 4(A), 2 ⁇ g/mL VHH-hFc was used to coat the ELISA plate, and the human HER2 protein was serially diluted from 30 ⁇ g/mL, and the EC80 was calculated as the concentration in the competitive ELISA.
  • VHH-hFc Dilute VHH-hFc with PBS to 2 ⁇ g/mL, coat a 96-well high-adsorption microtiter plate with 50 ⁇ L/well, and coat with 250 ⁇ L blocking solution (PBS containing 2% (w/w) BSA) at room temperature after overnight at 4°C. Blocking for two hours, adding 40 ⁇ g/mL of the antibody to be detected, then adding human HER2-his protein at the EC80 concentration corresponding to each antibody to be detected, giving it for 2 hours, washing with PBS for 5 times and adding HRP-labeled anti-His II Antibody, incubate for 1 hour, and wash the plate 5 times.
  • PBS containing 2% (w/w) BSA
  • VHH antibodies were classified according to the competition rate, and the results are shown in Figure 16.
  • Anti there is competition and are classified into one category; Compete, belong to one class; NB146-54, NB146-125, NB147-102, NB147-110 compete and belong to one class, NB146-27 does not compete with other antibodies, separate class; Tab050 (Pertuzumab) Anti-) and Tab094 (FRP5 mAb) compete with each other and belong to a class.

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Abstract

一种特异性结合HER2的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、宿主细胞、药物组合物、试剂盒、制备方法及其在治疗肿瘤或癌症以及检测HER2中的应用，对于HER2抗体治疗药物和检测试剂的开发具有重要意义。

Description

HER2抗体及其应用
本公开要求于2020年12月18日提交中国专利局、申请号为202011503677.9、发明名称为“HER2抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体领域，尤其涉及HER2抗体及其应用。
背景技术
HER2是由HER2/neu基因编码，位于细胞膜表面的受体酪氨酸激酶，属于表皮生长因子受体(EGFR)家族，通常与细胞生长和分化的信号传导有关。HER2没有天然配体，但可通过过表达同源二聚体或者通过与可被配体结合而活化的其他HER家族的成员的异源二聚体而激活，进而激活受体酪氨酸激酶，通过分裂原活化蛋白激酶(MAPK)、磷脂酰肌醇-3-激酶-蛋白激酶B/Akt(PI3K-PKB/Akt)、磷脂酶C-蛋白激酶C(PLC-PKC)及转录信号转导和活化蛋白(STAT)等多条信号通路触发下游信号的级联反应(Olayioye MA,Breast Cancer Res.2001；3(6):385-389；Huang等,Expert Opin Biol Ther.2009；9:97-110)。HER2在极少数组织内低水平表达，而在30％以上的人类肿瘤中过表达。HER2基因的过表达不仅与肿瘤的发生发展相关，还是一个重要的临床治疗检测与预后指标，并且是肿瘤靶向治疗药物选择的一个重要靶点(Cho HS and Leahy DJ,Science.2002；297(5585):1330-1333)。
曲妥珠单抗(商品名，Herceptin，赫赛汀)是1998年经美国FDA批准上市的重组人源化单克隆抗HER2抗体，其针对HER2蛋白胞外结构域IV，在过表达HER2的细胞中阻断配体非依赖性HER2同源二聚化，在更小的程度上与其他HER家族成员的异源二聚化(Cho等,Nature.2003；421:756-760；Wehrman等,Proc Natl Acad Sci USA.2006；103(50):19063-19068)。对于HER2过表达的原发性浸润性乳腺癌患者非常有效的曲妥珠单抗在高表达HER2的乳腺癌肿瘤上有效，但受限于HER2高表达且初始应答者有可能复发(Dinh等,Clin Adv Hematol Oncol.2007；5(9):707-717)。
帕妥珠单抗(商品名，Perjeta)是另有一种人源化单克隆抗HER2抗体。其针对HER2蛋白发生二聚化的结构域II，从而阻断HER2异源二聚体的形成(Hughes等,Mol Cancer Ther.2009；8(7):1885-1892)。帕妥珠单抗不严格地要求高水平的HER2表达，为HER2低表达乳腺癌患者提供更多治疗方案(Franklin等,Cancer Cell.2004；5(4):317-328)。帕妥珠单抗和曲妥珠单抗的联用可以增强抗肿瘤的效果(Baselga等,J Clin Oncol.2010；28:1138-1144)。
传统的单克隆抗体分子量大，组织渗透性差，治疗效果有限；鼠源单克隆抗体具有很高的免疫原性，而改造后的嵌合抗体和人源化抗体的亲和力成熟更具挑战性；全人源单克隆抗体的制备成本高、开发周期长、产出低等因素也限制了研发和推广。
1993年比利时科学家首次发现在骆驼血液中有一类缺失轻链的重链抗体(HCAbs)，该类抗体只包含一个重链可变区和两个常规的CH2和CH3区，但其具有很好的结构稳定性与抗原结合活性。重链抗体和由此衍生的VHH结构域具有分子量小、化学性质灵活、易表达、可溶性好、渗透性强、免疫原性弱、人源化简单、容易耦联其他分子等优点，弥补了传统抗 体缺陷的同时增加了药物开发的多样性。因此，本领域迫切需要开发新的有效针对HER2的特异性VHH结构域或重链抗体。
发明内容
本公开提供一种特异性结合HER2的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、宿主细胞、药物组合物、试剂盒、制备方法及其在治疗疾病以及检测HER2中的应用。
在第一方面，本发明涉及一种特异性结合HER2的抗体或抗原结合片段，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3。
在一些具体的实施方式中，根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定所述HCDR1、HCDR2和HCDR3；可选地，所述HCDR1、HCDR2和HCDR3选自表9；
在一些具体的实施方式中，所述HCDR1选自SEQ ID NO:34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、88、91、94、97、100、103、106、109、112、115、118、121、124、127、130、133、136、139、142、145、148、151、154、157、160、163、166、169、172、175、178、181、184、187、190或193；
在一些具体的实施方式中，所述HCDR2选自SEQ ID NO:35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、89、92、95、98、101、104、107、110、113、116、119、122、125、128、131、134、137、140、143、146、149、152、155、158、161、164、167、170、173、176、179、182、185、188、191或194；
在一些具体的实施方式中，所述HCDR3选自SEQ ID NO:36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、87、90、93、96、99、102、105、108、111、114、117、120、123、126、129、132、135、138、141、144、147、150、153、156、159、162、165、168、171、174、177、180、183、186、189、192或195。
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:34～36、SEQ ID NO：37～39或SEQ ID NO：40～42；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:43～45、SEQ ID NO：46～48或SEQ ID NO：49～51；
优先地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:52～54、SEQ ID NO：55～57或SEQ ID NO：58～60；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:61～63、SEQ ID NO：64～66或SEQ ID NO：67～69；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:70～72、SEQ ID NO：73～75或SEQ ID NO：76～78；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:79～81、SEQ ID NO：82～84或SEQ ID NO：85～87；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:88～90、SEQ ID NO:91～93或SEQ ID NO：94～96；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2 和HCDR3选自SEQ ID NO:97～99、SEQ ID NO：100～102或SEQ ID NO：103～105；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:106～108、SEQ ID NO：109～111或SEQ ID NO：112～114；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:115～117、SEQ ID NO：118～120或SEQ ID NO：121～123；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:124～126、SEQ ID NO：127～129或SEQ ID NO：130～132；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:133～135、SEQ ID NO：136～138或SEQ ID NO：139～141；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:142～144、SEQ ID NO：145～147或SEQ ID NO：148～150；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:151～153、SEQ ID NO：154～156或SEQ ID NO：157～159；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:160～162、SEQ ID NO：163～165或SEQ ID NO：166～168；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:169～171、SEQ ID NO：172～174或SEQ ID NO：175～177；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:178～180、SEQ ID NO：181～183或SEQ ID NO：184～186；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:187～189、SEQ ID NO：190～192或SEQ ID NO：193～195。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
在一些具体的实施方式中，所述单域抗体包含选自SEQ ID NO：16～33任一项所示的序列；可选地，所述单域抗体包含与SEQ ID NO：16～33任一项所示的序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；可选地，所述单域抗体包含与SEQ ID NO：16～33任一项所示的序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体包含SEQ ID NO：16～33任一项所示VHH结构域中的FR区；可选地，所述抗体包含与SEQ ID NO：16～33任一项所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；可选地，所述抗体包含与SEQ ID NO：16～33任一项所示VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些具体的实施方式中，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述抗体或抗原结合片段为重链抗体。
在一些具体的实施方式中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在一些具体的实施方式中，所述抗体或抗原结合片段特异性结合人HER2、猴HER2和/或鼠HER2，优选地，所述抗体或抗原结合片段与人HER2、猴HER2和鼠HER2的KD小于1E-6M、1E-7M、2E-7M、3E-7M、4E-7M、5E-7M、6E-7M、8E-7M、9E-7M、1E-8M、2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、8E-8M、9E-8M、；1E-9M、2E-9M、3E-9M、4E-9M、5E-9M、6E-9M、8E-9M、9E-9M、1E-10M或1E-11M。
在第二方面，本公开涉及一种特异性结合Her2的抗体或抗原结合片段，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别包含选自SEQ ID NO：16～33任一项所示VHH结构域的HCDR1、HCDR2和HCDR3。
在一些具体的实施方式中，根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定所述HCDR1、HCDR2和HCDR3；可选地，所述HCDR1、HCDR2和HCDR3选自表9；
可选地，所述HCDR1选自SEQ ID NO:34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、88、91、94、97、100、103、106、109、112、115、118、121、124、127、130、133、136、139、142、145、148、151、154、157、160、163、166、169、172、175、178、181、184、187、190或193；
可选地，所述HCDR2选自SEQ ID NO:35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、89、92、95、98、101、104、107、110、113、116、119、122、125、128、131、134、137、140、143、146、149、152、155、158、161、164、167、170、173、176、179、182、185、188、191或194；
可选地，所述HCDR3选自SEQ ID NO:36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、87、90、93、96、99、102、105、108、111、114、117、120、123、126、129、132、135、138、141、144、147、150、153、156、159、162、165、168、171、 174、177、180、183、186、189、192或195；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:34～36、SEQ ID NO：37～39或SEQ ID NO：40～42；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:43～45、SEQ ID NO：46～48或SEQ ID NO：49～51；
优先地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:52～54、SEQ ID NO：55～57或SEQ ID NO：58～60；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:61～63、SEQ ID NO：64～66或SEQ ID NO：67～69；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:70～72、SEQ ID NO：73～75或SEQ ID NO：76～78；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:79～81、SEQ ID NO：82～84或SEQ ID NO：85～87；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:88～90、SEQ ID NO:91～93或SEQ ID NO：94～96；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:97～99、SEQ ID NO：100～102或SEQ ID NO：103～105；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:106～108、SEQ ID NO：109～111或SEQ ID NO：112～114；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:115～117、SEQ ID NO：118～120或SEQ ID NO：121～123；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:124～126、SEQ ID NO：127～129或SEQ ID NO：130～132；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:133～135、SEQ ID NO：136～138或SEQ ID NO：139～141；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:142～144、SEQ ID NO：145～147或SEQ ID NO：148～150；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:151～153、SEQ ID NO：154～156或SEQ ID NO：157～159；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:160～162、SEQ ID NO：163～165或SEQ ID NO：166～168；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:169～171、SEQ ID NO：172～174或SEQ ID NO：175～177；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:178～180、SEQ ID NO：181～183或SEQ ID NO：184～186；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:187～189、SEQ ID NO：190～192或SEQ ID NO：193～195。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2 和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段与曲妥珠单抗、帕妥珠单抗或FRP5单抗竞争性结合HER2，或所述抗体或抗原结合片段不与曲妥珠单抗、帕妥珠单抗或FRP5单抗竞争性结合HER2。
在一些具体的实施方式中，所述抗体或抗原结合片段还连接有其他功能性分子，优选地，所述其他功能性分子可选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂或免疫检查点抑制剂。
在一些具体的实施方式中，所述细胞因子可为IL2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha；所述血管生成抑制剂可为内皮抑制素；所述免疫检查点抑制剂可为SIRPα。
在第三方面，本公开还公开一种多特异性抗原结合分子，所述多特异性抗原结合分子包含前述的抗体或抗原结合片段，以及结合HER2以外其他抗原的抗原结合分子，或结合与前述抗体或抗原结合片段不同的HER2表位；可选地，所述HER2以外的其他抗原可选自：CD3，优选CD3ε；CD16，优选CD16A；CD137；CD258；4-1BB；CD40；CD64；EGFR；HER1；HER3；PD-1；PD-L1；VEGF；IGF-IR(胰岛素样生长因子I型受体)；磷脂酰丝氨酸(Phosphatidylserine，PS)；c-Met或血脑屏障受体；
优选地，所述其他抗原结合分子为抗体或抗原结合片段；
优选地，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性；
优选地，所述多特异性抗原结合分子可为二价、四价或六价。
在第四方面，本公开还公开一种嵌合抗原受体，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含前述抗体或抗原结合片段。
在第五方面，本公开还公开一种免疫效应细胞，所述免疫效应细胞表达前述的嵌合抗原受体，或包含编码前述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
在第六方面，本公开还公开一种分离的核酸片段，所述核酸片段编码前述抗体或抗原结合片段、多特异性抗原结合分子或嵌合抗原受体。
在第七方面，本公开还公开一种载体(vector)，所述载体包含前述的核酸片段。
在第八方面，本公开还公开一种宿主细胞，所述宿主细胞包含前述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在第九方面，本公开还公开一种制备前述抗体或抗原结合片段或多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在第十方面，本公开还公开一种制备前述免疫效应细胞的方法，所述方法包括将编码前述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达前述CAR。
在第十一方面，本公开还公开一种药物组合物，所述药物组合物包含前述的抗体或抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体或根据前述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
在第十二方面，本公开还公开一种治疗肿瘤或癌症的方法，所述方法包括向受试者施用有效量的前述的抗体或抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
在第十三方面，本公开还公开前述抗体或抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物在制备治疗肿瘤或癌症药物中的用途；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
在第十四方面，本公开还公开前述的抗体或抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物，用于治疗肿瘤或癌症；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
在第十五方面，本公开还公开一种试剂盒，所述试剂盒包含前述的抗体或抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物。
在第十六方面，本公开还公开一种检测生物学样品中HER2表达的方法，所述方法包括在前述的抗体或抗原结合片段与HER2之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中HER2的存在或表达水平。
在第十七方面，本公开还公开前述抗体或抗原结合片段在制备HER2检测试剂中的用途。
术语定义
除非本文另外定义，与本公开相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“Her2”：也被称为ErbB2、NEU或CD340，其包括人表皮生长因子受体2(UniProtKB ID:P04626-1)及其突变体(variants)、同种型(isoforms)和物种同源物(species homologs)。本文HER2的物种同源物包括但不限于脊椎动物或哺乳动物的HER2及其突变体和同种型，例如，猴Her2(NCBI ID:XP_014975023.1)和小鼠Her2。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10 -6M或更低、10 -7M或更低、约10 -8M或更低、约1×10 -9M或更低、约1×10 -10M或更低、1×10 -11M或更低或1×10 -12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是 在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
本文术语“VHH结构域”和“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“单域抗体”、“VHH结构域”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。示例性地，本文“抗原结合片段”或“抗体片段”包括但不限于Fab、F(ab’)2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠、小鼠、兔或羊驼)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985 J Immunol Methods 125:191-202；以上通过援引加入并入本文。
本文术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力或增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网 站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”可选自CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文中的术语“Fc区”用于定义抗体重链中含有恒定区的至少一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。示例性地，人IgG重链Fc区可自Cys226或Pro230延伸至重链的羧基末端。然而，由宿主细胞生成的抗体可经历翻译后切割，自重链的C端切除一个或多个，特别是一个或两个氨基酸。因此，通过编码全长重链的特定核酸分子的表达由宿主细胞生成的抗体可包括全长重链，或者它可包括全长重链的切割变体。当重链的最终两个C端氨基酸是甘氨酸(G446)和赖氨酸(K447，编号方式依照Kabat EU索引)时可能就是这种情况。因此，Fc区的C端赖氨酸(Lys447)，或C端甘氨酸(Gly446)和赖氨酸(Lys447)可以存在或不存在。
典型地，IgG Fc区包含IgG CH2和IgG CH3域，可选地，在此基础上还可包含完整或部分铰链区，但不包含CH1域。人IgG Fc区的“CH2域”通常自约位置231处的氨基酸残基延伸至约位置340处的氨基酸残基。在一个实施方案中，碳水化合物链附着于CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中在CH2域C端的那段残基(即自IgG的约位置341处的氨基酸残基至约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域(例如具有在其一条链中引入的“隆起”(“节”，knob)和在其另一条链中相应引入的“空腔”(“穴”，hole)的CH3域；参见美国专利No.5,821,333， 通过援引明确收入本文)。如本文中描述的，此类变体CH3域可用于促进两条不相同抗体重链的异二聚化。
除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号依照EU编号系统，也称作EU索引，如Kabat et al.,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,Bethesda,MD,1991中描述的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本公开所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本公开核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本公开蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比 对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其至少包含(1)细胞外抗原结合结构域，例如抗体的重链可变区和/或轻链可变区，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本公开的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2101823B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾 病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本文所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
附图说明
图1A为ELISA检测对照抗体与人HER2-His蛋白的结合反应；
图1B为ELISA检测对照抗体与猴HER2-His蛋白的结合反应；
图2A-2D为对照抗体检测SK-BR-3细胞HER2表达量的FACS结果；
图3A-3F为人HER2蛋白转染的CHO-K1细胞FACS筛选检测结果；
图4为Tab048抗体检测HEK293T-猴HER2细胞HER2表达量的FACS结果；
图5A为ELISA检测人HER2蛋白免疫后羊驼血清抗体效价情况；
图5B为FACS检测人HER2蛋白免疫后羊驼血清抗体效价情况；
图6A-6C为ELISA检测VHH-hFc与人HER2-His蛋白的结合反应；
图7A-7C为ELISA检测VHH-hFc与人HER2-CHO-K1细胞的结合反应；
图8A-8C为ELISA检测VHH-hFc与SK-BR-3肿瘤细胞的结合反应；
图9A-9C为ELISA检测VHH-hFc与猴HER2-His蛋白的结合反应；
图10A-10C为ELISA检测VHH-hFc与鼠HER2-His蛋白的结合反应；
图11A-11C为ELISA检测VHH-hFc与HEK293T-猴HER2细胞的结合反应；
图12A-12B为SPR检测VHH-hFc与人HER2的亲和力；
图13A-13B为SPR检测VHH-hFc与猴HER2的亲和力；
图14为SPR检测VHH-hFc与鼠HER2的亲和力；
图15为竞争性ELISA方法检测VHH-hFc之间的抑制率；
图16 VHH-hFc的抗原表位分类。
具体实施方式
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
实施例1对照抗体制备、内源细胞鉴定和过表达细胞株的制备
(A)、对照抗体的制备
将识别人HER2的单克隆抗体FRP5、4D5(曲妥珠单抗)和2C4(帕妥珠单抗)VH和VL序列重组到人IgG1 CH和CL表达载体(其中表达载体购自Biointron，重组步骤也由Biointron完成)中，得重组质粒(上述质粒重组的实验原理及步骤的出处见《分子克隆实验指南(第三版)》，(美)J.萨姆布鲁克等著)。将克隆FRP5的VH和VL通过3个GGGGS连接子连接后重组到人IgG1 Fc的表达载体中，得到重组质粒，并经测序验证。使用碱裂解法试剂盒(购自QIAGEN)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。以上质粒构建及抗体的表达纯化工作均由泰州市百英生物科技有限公司完成。
在培养基293medium(培养基A，购自Biointron)培养293F细胞(购自Invitrogen)。摇床设置为37℃、120RPM和8％CO 2(v/v)。取2mL培养基A和120μl 1μg/mL PEI(购自Biointron)混匀，得培养基B。取2mL培养基A和30μg重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将4mL混合液D缓缓加入60mL含293F细胞的培养基293medium中至293F的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。连续培养5天，收集上清，收集上清，8000rpm离心5min，丢弃细胞，上清以供纯化。
对于连续生产的无内毒素的层析柱和蛋白A填料，使用0.1M NaOH处理30min或者5个柱体积0.5M NaOH冲洗；对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒素的水冲洗至中性，用10倍柱体积的1％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将离心好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积PBS清洗。用5倍柱体积的pH3.4的柠檬酸缓冲液进行洗脱，收集洗脱液，并用1/10体积的pH8.0的1M Tris中和。收获抗体后，在1×PBS中透析过夜，避免内毒素污染。透析结束后，使用Nanodrop测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自安度斯)检测抗体内毒素含量。
将FRP5人IgG1形式的抗体、4D5人IgG1形式的抗体、2C4人IgG1形式的抗体和FRP5 ScFv-人IgG1 Fc(hFc)形式的抗体分别命名为Tab048，Tab049，Tab050和Tab094，具体序列信息如表1所示。
表1对照抗体的序列信息
Figure PCTCN2021139018-appb-000001
Figure PCTCN2021139018-appb-000002
Figure PCTCN2021139018-appb-000003
通过ELISA方法检测对照抗体与人HER2-His蛋白(购自Acro，货号：HE2-H5225)以及猴HER2-His蛋白(购自Sino Biological，货号：90295-C08H)的结合活性，具体检测结果如表2-3和图1A-1B所示，其中IgG亚型对照为hIgG1，图表数据为OD 450值。结果表明，Tab048、Tab049、Tab050、Tab094抗体与人HER2蛋白以及猴HER2蛋白有很好的结合活性。
表2 ELISA检测对照抗体与人HER2-his蛋白的结合反应
抗体浓度(nM) Tab048 Tab049 Tab050 hIgG1 抗体浓度(nM) Tab094
100 1.87 2.38 2.30 0.11 200 2.32
10 2.22 2.22 2.37 0.06 20 2.36
1 2.36 2.31 2.45 0.06 2 2.51
0.1 1.95 1.97 1.90 0.05 0.2 2.27
0.01 0.72 0.74 0.59 0.07 0.02 1.72
0.001 0.13 0.14 0.17 0.06 0.002 0.40
0.0001 0.06 0.06 0.06 0.06 0.0002 0.12
0.00001 0.05 0.05 0.06 0.05 0.00002 0.07
表3 ELISA检测对照抗体与猴HER2-his蛋白的结合反应
抗体浓度(nM) Tab048 Tab049 Tab050 hIgG1 抗体浓度(nM) Tab094
100 2.35 2.35 2.42 0.12 200 2.38
10 2.04 2.07 2.15 0.07 20 2.23
1 2.24 2.39 2.04 0.11 2 2.30
0.1 1.83 2.07 1.11 0.05 0.2 2.17
0.01 0.52 0.79 0.16 0.08 0.02 1.49
0.001 0.11 0.14 0.06 0.05 0.002 0.36
0.0001 0.05 0.06 0.06 0.06 0.0002 0.09
0.00001 0.05 0.05 0.05 0.05 0.00002 0.05
(B)、内源性表达HER2蛋白的细胞株鉴定
将SK-BR-3细胞在T-75细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用Tab048、Tab049、Tab050和Tab094抗体作为一抗，APC标记的 二抗(购自Biolegend，货号：409306)经FACS(FACS CantoTM，购自BD公司)检测和分析。结果如表4以及图2A-2D所示，说明SK-BR-3细胞与Tab048、Tab049、Tab050和Tab094均可结合。
表4 FACS检测SK-BR-3细胞HER2表达量
Figure PCTCN2021139018-appb-000004
(C)、表达人HER2蛋白的CHO-K1重组细胞株的制备
编码人HER2全长氨基酸序列(UniProtKB ID:P04626-1，网址：https://www.uniprot.org/uniprot/P04626，isoform1)的核苷酸序列被克隆到pcDNA3.1载体(购自Clontech)并制备质粒。对CHO-K1细胞系(购自中国科学院典型培养物保藏委员会细胞库)进行质粒转染(
Figure PCTCN2021139018-appb-000005
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/mL puromycin的含10％(w/w)胎牛血清的DMEM/F12培养基中选择性培养2周，流式细胞仪检测表达量。有限稀释法挑选高表达的单克隆，即调整细胞密度为0.5个每孔后种到96孔板，置于37℃，5％(v/v)CO 2培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存。具体选择结果如表5和图3A-3F所示，仅孵育二抗的作为对照。表5的检测结果表明，已经制得一系列人HER2阳性表达的CHO-K1单克隆细胞系。图3A-3F横坐标为细胞荧光强度，纵坐标为细胞数。结果说明，1B8(13)、3D8(32)、1F7(11)、3D10(34)、2F9(21)、1C4(6)为人HER2高水平表达细胞株，后续筛选选用1F7(11)克隆。
表5表达人HER2蛋白的CHO-K1重组细胞系FACS检测结果
Figure PCTCN2021139018-appb-000006
(D)、表达猴HER2蛋白的重组HEK293T细胞株制备
编码猴HER2全长氨基酸序列(NCBI ID:XP_014975023.1，网址https://www.ncbi.nlm.nih.gov/protein/XP_014975023.1/)的核苷酸序列被克隆到pcDNA3.1载体(购自Thermofisher scientific)并制备质粒。对HEK293T细胞系用
Figure PCTCN2021139018-appb-000007
HD(Promega，货号：#E2311)进行质粒转染后，在含5μg/mL puromycin的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分多克隆孔扩增到6孔板中。对扩增后的克隆用Tab048抗体经 FACS流式细胞仪检测和分析，选择长势较好、荧光强度较高的细胞株继续扩大培养并液氮冻存。表达量的结果如图4，显示经过puromycin加压筛选后的HEK293T-猴-HER2具有单一的阳性峰，可用于检测抗体的交叉活性。
实施例2针对HER2的单域抗体VHH的制备
(A)、羊驼免疫及血清效价检测
采用人HER2(Thr 23-Thr 652)-His蛋白(购自Acro，货号：HE2-H82E2)免疫两只羊驼(Alpaca，编号为NB146和NB147)。初次免疫时，人HER2-His蛋白用弗氏完全佐剂乳化后皮下多点注射，即每只羊驼注射500μg人HER2-His蛋白。加强免疫时，人HER2-His蛋白用弗氏不完全佐剂乳化后皮下多点注射，即每只羊驼注射250μg人HER2-His蛋白。初次免疫与第一次加强免疫之间间隔3周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中人HER2-His抗体的效价和特异性，结果如图5A-5B和表6-7所示。结果表明，经人HER2-His蛋白免疫后的羊驼血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中ELISA最高稀释度在24300左右。空白对照为1％(w/w)BSA，批次指第三次和第四次加强免疫后第七天的羊驼血清TB2和TB3，表中的数据为OD 450nm值。
表6 ELISA检测人HER2-His蛋白免疫后的羊驼血清抗体效价
Figure PCTCN2021139018-appb-000008
表7 FACS检测人HER2蛋白免疫后的羊驼血清抗体效价
Figure PCTCN2021139018-appb-000009
Figure PCTCN2021139018-appb-000010
(B)、噬菌体文库构建及针对HER2纳米抗体淘选
四次蛋白免疫后一周，采集100mL羊驼外周血，使用淋巴分离液分离PBMC，并使用RNAiso Plus试剂提取总RNA。使用PrimeScript TM II 1st Strand cDNA Synthesis Kit(购自Takara，货号：6210A)将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体可变区的核酸片段：
第一轮PCR：
上游引物：CTTGGTGGTCCTGGCTGC(SEQ ID NO：11)；
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO：12)。
第二轮PCR：
以第一轮PCR产物作为模板，
上游引物：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC(SEQ ID NO：13)；
下游引物-1：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG(SEQ ID NO：14)；
下游引物-2：CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG(SEQ ID NO：15)。
回收目标单域抗体核酸片段，并使用限制性内切酶SfiI将其克隆进入噬菌体展示用载体pcomb3XSS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对HER2的单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为3.4×10 9。为检测文库的插入率，随机选取48个克隆做菌落PCR。结果显示插入率达到100％。
(C)、针对HER2的单域抗体淘选
将人HER2-His蛋白用pH值为9.6的碳酸盐缓冲液稀释至终浓度为5μg/mL，按100μL/孔加入酶标孔中，每个蛋白包被8孔，4℃包被过夜；弃包被液，PBS洗涤3次，每孔加入300μL 3％BSA-PBS封闭液，37℃封闭1小时；PBS洗涤3次，加入100μL噬菌体文库，37℃孵育1小时；吸出未结合的噬菌体，用PBST洗涤6次，PBS洗涤2次；加入100μL Gly-HCl洗脱液，37℃孵育8分钟，洗脱特异性结合的噬菌体；将该洗脱液转移至1.5mL无菌离心管中，迅速用10μL Tris-HCl中和缓冲液中和；取10μL进行梯度稀释，测定滴度，计算淘选回收率，其余洗脱物混合后进行扩增和纯化，可用于下一轮亲和淘选。
从第一轮淘选洗脱物滴度的平板上，用灭菌牙签随机挑取192个单克隆接种于1mL 2×YT-AK中，37℃，220r/min振荡培养8小时。取100μL上述培养物，按cell：phage＝1：20的比例加入M13K07噬菌体，37℃，静置15min，220r/min振荡培养45分钟。补加300μL体积的2×YT-AK，30℃，剧烈振荡培养过夜。第二天12000rpm离心2分钟，取上清，用于单克隆ELISA鉴定。
将人HER2蛋白和猴HER2蛋白用pH值为9.6的碳酸盐缓冲液稀释至终浓度为2μg/mL和1μg/mL，按100μL孔加入酶标孔中，4℃包被过夜；弃包被液，PBST洗涤3次，每孔加入300μL 5％脱脂牛奶，37℃封闭1小时；PBST洗涤3次，每孔加入50μL噬菌体培养菌液上清和50μL 5％脱脂牛奶，37℃，孵育1小时；PBST洗涤5次，加入辣根过氧化物酶标记的抗M13抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1小时；PBST洗板6次。 加入TMB显色液显色，100μL/孔，37℃，7分钟，加入终止液终止反应，50μL/孔，于450nm波长下测光密度。挑选人HER2阳性，人HER2和猴HER2双阳性克隆送成都擎科梓熙生物技术有限公司进行测序。
对测序结果进行分析，根据VHH编码蛋白序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列，选择以下VHH抗体(详见表8)，采用生物信息学方法分析其CDR区，具体结果如表9所示，并进行后续的VHH-hFc生产和鉴定。分析方式包括：Chothia definition和Kabat definition，使用如下分析网站：http://cao.labshare.cn/AbRSA/abrsa.php，http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi；IMGT definition，使用如下分析网站：http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。
表8 VHH抗体序列信息
Figure PCTCN2021139018-appb-000011
Figure PCTCN2021139018-appb-000012
表9抗体CDR区信息
Figure PCTCN2021139018-appb-000013
Figure PCTCN2021139018-appb-000014
Figure PCTCN2021139018-appb-000015
实施例3 VHH-hFc生产
将目标的VHH序列重组到人IgG1 Fc的表达载体中，得重组质粒。具体的质粒构建、转染以及纯化流程参照实施例1(A)。
将纯化的VHH-hFc进行蛋白浓度、纯度、内毒(Lonza试剂盒)等检测分析，结果如表10所示，结果发现，抗体最终产品纯度较高，内毒素浓度在1.0EU/mg以内。
表10纯化的VHH-hFc抗体质量控制
抗体名称 克隆号 抗体浓度(mg/mL) 抗体纯度(SEC，280nm)％ 内毒素EU/mg
NB146-171 NB146-171 0.85 99.23 <1
NB146-27 NB146-27 0.31 99.47 <1
NB146-62 NB146-62 0.75 93.18 <1
NB146-12 NB146-12 0.42 92.06 <1
NB146-54 NB146-54 0.88 99.01 <1
NB146-28 NB146-28 0.73 99.69 <1
NB146-125 NB146-125 0.96 99.60 <1
NB147-83 NB147-83 0.97 92.80 <1
NB147-102 NB147-102 0.69 98.92 <1
NB147-110 NB147-110 0.73 98.54 <1
NB147-137 NB147-137 0.94 98.49 <1
NB147-14 NB147-14 0.76 90.48 <1
NB147-161 NB147-161 0.87 98.88 <1
NB147-32 NB147-32 1.58 99.29 <1
NB147-36 NB147-36 2.96 89.42 <1
NB147-39 NB147-39 2.46 98.87 <1
NB147-61 NB147-61 2.52 98.71 <1
NB147-66 NB147-66 1.62 97.52 <1
实施例4 VHH-hFc的鉴定
(A)、酶联免疫吸附实验(ELISA)检测VHH-hFc与人HER2蛋白的结合
人HER2蛋白用PBS稀释到终浓度2μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入100nM梯度稀释的VHH-hFc或对照抗体50μl每孔。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma，货号：A0170)，37℃孵育1小时后，用PBS洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD 450nm数值，VHH-Fc与人HER2蛋白的结合活性结果如图6A-6C和表11所示，说明纯化后的抗体与人HER2蛋白均有结合。其中IgG对照为hIgG1，表中的数据为OD 450nm值，N/A表示此处无数据。
表11 ELISA检测VHH-hFc与人HER2蛋白的结合反应
Figure PCTCN2021139018-appb-000016
Figure PCTCN2021139018-appb-000017
(B)、流式细胞实验(FACS)检测抗体与表达HER2蛋白的细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，吸除培养基，用PBS缓冲液洗涤2次，用胰酶消化细胞，然后用完全培养基终止消化，并吹打细胞至单细胞悬液。细胞计数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2x10 6细胞每毫升，按每孔50μl加入到96孔FACS反应板中，加入VHH-hFc或对照抗体每孔50μl，4℃孵育1小时。用PBS缓冲液离心洗涤3次，加入每孔50μl FITC标记的二抗(购自Invitrogen，货号：A18830)，冰上孵育1小时。用PBS缓冲液离心洗涤3次，100μl用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(FlowJo)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50。分析结果如表12以及图7A-7C(VHH-hFc与CHO-K1-人HER2)，图8A-8C(VHH-hFc与SK-BR-3)所示，表明VHH-hFc均可结合CHO-K1-人HER2细胞和SK-BR-3细胞。
表12 FACS检测VHH-hFc与表达人HER2的细胞结合反应
Figure PCTCN2021139018-appb-000018
Figure PCTCN2021139018-appb-000019
实施例5 VHH-hFc的交叉结合活性检测
(A)、ELISA检测VHH-hFc与猴HER2蛋白和鼠HER2蛋白的结合
将猴HER2-His蛋白(购自Sino Biological，货号：90295-C08H)和鼠HER2-his蛋白(购自Sino Biological，货号：50714-M08H)按照实施例4(A)的方法进行ELISA检测与数据分析。分析结果如图9A-9C、图10A-10C和表13-14所示，VHH-hFc均能与猴HER2蛋白结合，7个VHH-hFc能与鼠HER2蛋白结合。其中IgG对照为hIgG1，表中数据为OD 450nm值，N/A表示此处无数据。
表13 ELISA检测VHH-hFc与猴HER2蛋白的结合反应
Figure PCTCN2021139018-appb-000020
Figure PCTCN2021139018-appb-000021
表14 ELISA检测VHH-hFc与鼠HER2蛋白的结合反应
Figure PCTCN2021139018-appb-000022
Figure PCTCN2021139018-appb-000023
(B)ELISA检测VHH-hFc与表达猴HER2蛋白的细胞结合
将HEK293T-猴HER2细胞按照实施例4(B)的方法进行FACS检测与数据分析。分析结果如表15以及图11A-11C所示，所有VHH-hFc均与HEK293T-猴-HER2细胞有结合活性。
表15 FACS检测VHH-hFc与表达猴HER2蛋白的细胞结合反应
Figure PCTCN2021139018-appb-000024
实施例6 HER2-hFc的亲和力检测
(A)、VHH-hFc与人HER2蛋白亲和力检测
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗人HER2 VHH-hFc抗体。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然 后注入单一浓度的HER2抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的人HER2-His持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1：1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1：1模型。VHH-hFc与人HER2蛋白的结合速率(K a)、解离速率(K dis)及结合亲和力(KD)如表所示，其中抗体Tab094作为对照。如表16和图12A-12B所示，VHH-hFc与人HER2蛋白均有较好的亲和力，其中亲和力最低在1E-07M左右，亲和力最优在6E-09M左右。
表16 VHH-hFc与人HER2蛋白的结合亲和力
抗体名称 K a(1/Ms) K dis(1/s) KD(M)
NB146-125 6.82E+04 7.89E-04 1.16E-08
NB147-36 1.14E+05 6.39E-03 5.61E-08
NB147-39 1.12E+05 3.57E-03 3.18E-08
NB147-83 3.01E+04 2.08E-03 6.90E-08
NB146-171 3.81E+07 2.91E+00 7.64E-08
NB147-61 9.53E+04 1.08E-02 1.13E-07
NB147-102 2.23E+05 3.61E-03 1.62E-08
NB146-27 2.08E+04 8.78E-03 4.23E-07
NB147-66 2.19E+04 2.04E-03 9.30E-08
NB147-110 5.59E+04 4.38E-03 7.85E-08
NB146-62 2.62E+04 1.09E-02 4.18E-07
NB147-137 5.41E+04 1.44E-02 2.67E-07
NB146-12 2.61E+05 1.45E-02 5.54E-08
NB147-14 6.71E+05 1.99E-02 2.97E-08
NB146-54 4.83E+04 3.01E-04 6.22E-09
NB147-161 9.37E+04 4.65E-03 4.96E-08
NB146-28 1.94E+05 2.20E-03 1.13E-08
NB147-32 2.91E+04 2.00E-03 6.89E-08
Tab048 2.21E+05 7.45E-05 3.37E-10
Tab094 9.96E+04 4.57E-05 4.59E-10
(B)、HER2 VHH-hFc与猴HER2-his蛋白亲和力检测
按照实施例6(A)的方法对VHH-hFc与猴HER2-His蛋白进行亲和力检测，其中抗体Tab048、Tab094作为对照。如表17和图13A-13B所示，除NB146-27、NB146-62、NB147-137和NB147-161以外，VHH-hFc与猴HER2蛋白均具有较好的亲和力。
表17 VHH-hFc与猴HER2蛋白的结合亲和力
Figure PCTCN2021139018-appb-000025
(C)、HER2 VHH-hFc与鼠HER2-his蛋白亲和力检测
按照实施例6(A)的方法对VHH-hFc与鼠HER2-His蛋白进行亲和力检测，其中抗体Tab094作为对照。如表18和图14所示，NB147-32、NB147-36、NB147-66和NB147-61，与鼠HER2蛋白具有较好的亲和力。
表18 VHH-hFc与鼠HER2蛋白的结合亲和力
Figure PCTCN2021139018-appb-000026
Figure PCTCN2021139018-appb-000027
实施例7抗体抗原结合表位竞争实验(epitope binning)
为了鉴定抗体对抗原的结合位点，采用竞争ELISA的方法对HER2 VHH-hFc进行分组。按照实施例4(A)的方法使用2μg/mL VHH-hFc包被ELISA板，人HER2蛋白从30μg/mL开始进行梯度稀释，计算出EC80作为竞争性ELISA中的浓度。
用PBS稀释VHH-hFc至2μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250μL封闭液(含有2％(w/w)BSA的PBS)进行室温两小时封闭，加入40μg/mL待检测的抗体后，再加入每个待检测抗体对应的EC80浓度的人HER2-his蛋白，赋予2小时，用PBS洗5次后加入HRP标记的anti-His二抗，孵育1小时，洗板5次。加入TMB底物50μL每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μL每孔。用ELISA读板机(Insight，购自PerkinElmer)读取OD450nm数值，根据OD450nm数值，利用公式(抑制率＝(阴性对照OD450nm数值-竞争抗体样本OD450nm数值)*100％/阴性对照OD450nm数值计算出抗体相互之间的竞争率，结果如图15所示，竞争率的数值越高，表示两个抗体结合的抗原表面越是接近。
根据竞争率将VHH抗体进行分类，结果如图16所示，NB146-28，NB146-62，NB147-14，NB146-12，NB147-171，NB147-83，NB147-39与Tab049(曲妥珠单抗)存在竞争，归为一类；NB146-28，NB146-62，NB147-14，NB146-12，NB147-137，NB147-161，NB147-32，NB147-36，NB147-61，NB147-66相互竞争，属于一类；NB146-54，NB146-125，NB147-102，NB147-110之间存在竞争，归为一类，NB146-27不与其他抗体竞争，单独一类；Tab050(帕妥珠单抗)和Tab094(FRP5单抗)相互竞争，属于一类。

Claims (29)

  1. 一种特异性结合Her2的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别包含选自SEQ ID NO：16～33任一项所示VHH结构域的HCDR1、HCDR2和HCDR3。
  2. 根据权利要求1所述的抗体或抗原结合片段，其中，根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定所述HCDR1、HCDR2和HCDR3；可选地，所述HCDR1、HCDR2和HCDR3选自表9；
    可选地，所述HCDR1选自SEQ ID NO:34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、88、91、94、97、100、103、106、109、112、115、118、121、124、127、130、133、136、139、142、145、148、151、154、157、160、163、166、169、172、175、178、181、184、187、190或193；
    可选地，所述HCDR2选自SEQ ID NO:35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、89、92、95、98、101、104、107、110、113、116、119、122、125、128、131、134、137、140、143、146、149、152、155、158、161、164、167、170、173、176、179、182、185、188、191或194；
    可选地，所述HCDR3选自SEQ ID NO:36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、87、90、93、96、99、102、105、108、111、114、117、120、123、126、129、132、135、138、141、144、147、150、153、156、159、162、165、168、171、174、177、180、183、186、189、192或195；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:34～36、SEQ ID NO：37～39或SEQ ID NO：40～42；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:43～45、SEQ ID NO：46～48或SEQ ID NO：49～51；
    优先地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:52～54、SEQ ID NO：55～57或SEQ ID NO：58～60；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:61～63、SEQ ID NO：64～66或SEQ ID NO：67～69；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:70～72、SEQ ID NO：73～75或SEQ ID NO：76～78；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:79～81、SEQ ID NO：82～84或SEQ ID NO：85～87；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:88～90、SEQ ID NO:91～93或SEQ ID NO：94～96；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:97～99、SEQ ID NO：100～102或SEQ ID NO：103～105；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:106～108、SEQ ID NO：109～111或SEQ ID NO：112～114；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2 和HCDR3选自SEQ ID NO:115～117、SEQ ID NO：118～120或SEQ ID NO：121～123；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:124～126、SEQ ID NO：127～129或SEQ ID NO：130～132；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:133～135、SEQ ID NO：136～138或SEQ ID NO：139～141；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:142～144、SEQ ID NO：145～147或SEQ ID NO：148～150；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:151～153、SEQ ID NO：154～156或SEQ ID NO：157～159；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:160～162、SEQ ID NO：163～165或SEQ ID NO：166～168；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:169～171、SEQ ID NO：172～174或SEQ ID NO：175～177；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:178～180、SEQ ID NO：181～183或SEQ ID NO：184～186；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:187～189、SEQ ID NO：190～192或SEQ ID NO：193～195。
  3. 根据权利要求1或2所述的抗体或抗原片段，其中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  4. 根据权利要求1或2所述的抗体或抗原结合片段，其中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  5. 根据权利要求1～4所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
  6. 根据权利要求5所述的抗体或抗原结合片段，其中，所述单域抗体包含选自SEQ ID NO：16～33任一项所示的序列；
    可选地，所述单域抗体包含与SEQ ID NO：16～33任一项所示的序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述单域抗体包含与SEQ ID NO：16～33任一项所示的序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  7. 根据权利要求1～6任一项所述的抗体或抗原结合片段，其中，所述抗体包含SEQ ID NO：16～33任一项所示VHH结构域中的FR区；
    可选地，所述抗体包含与SEQ ID NO：16～33任一项所示VHH结构域中的FR区相比发 生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述抗体包含与SEQ ID NO：16～33任一项所示VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  8. 根据权利要求1～7任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  9. 根据权利要求1～8任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区选自IgG、IgM、IgA、IgE或IgD，所述IgG选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述抗体或抗原结合片段为重链抗体。
  10. 根据权利要求1～9任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  11. 根据权利要求1～10任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段特异性结合人HER2、猴HER2和/或鼠HER2，优选地，所述抗体或抗原结合片段与人HER2、猴HER2和鼠HER2的KD小于1E-6M、1E-7M、2E-7M、3E-7M、4E-7M、5E-7M、6E-7M、8E-7M、9E-7M、1E-8M、2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、8E-8M、9E-8M、；1E-9M、2E-9M、3E-9M、4E-9M、5E-9M、6E-9M、8E-9M、9E-9M、1E-10M或1E-11M。
  12. 根据权利要求1～11任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段与曲妥珠单抗、帕妥珠单抗或FRP5单抗竞争性结合HER2，或所述抗体或抗原结合片段不与曲妥珠单抗、帕妥珠单抗或FRP5单抗竞争性结合HER2。
  13. 根据权利要求1～12任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段还连接有其他功能性分子，优选地，所述其他功能性分子选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂或免疫检查点抑制剂。
  14. 根据权利要求13所述的抗体或抗原结合片段，其中，所述细胞因子为IL2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha；所述血管生成抑制剂为内皮抑制素；所述免疫检查点抑制剂为SIRPα。
  15. 一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子包含权利要求1～14任一项所述的抗体或抗原结合片段，以及结合HER2以外其他抗原的抗原结合分子，或结合与权利要求1～14任一项所述抗体或抗原结合片段不同的HER2表位；可选地，所述HER2以外的其他抗原选自：CD3，优选CD3ε；CD16，优选CD16A；CD137；CD258；4-1BB；CD40；CD64；EGFR；HER1；HER3；PD-1；PD-L1；VEGF；IGF-IR(胰岛素样生长因子I型受体)；磷脂酰丝氨酸(Phosphatidylserine，PS)；c-Met或血脑屏障受体；
    优选地，所述其他抗原结合分子为抗体或抗原结合片段；
    优选地，所述多特异性抗原结合分子为双特异性、三特异性或四特异性；
    优选地，所述多特异性抗原结合分子为二价、四价或六价。
  16. 一种嵌合抗原受体，其中，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1～14任一项所述抗体或抗原结合片段。
  17. 一种免疫效应细胞，其中，所述免疫效应细胞表达权利要求16所述的嵌合抗原受体，或包含编码权利要求16所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  18. 一种分离的核酸片段，其中，所述核酸片段编码权利要求1～14任一项所述抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子或权利要求16所述嵌合抗原受体。
  19. 一种载体(vector)，其中，所述载体包含权利要求18所述的核酸片段。
  20. 一种宿主细胞，其中，所述宿主细胞包含权利要求19所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
  21. 一种制备权利要求1～14任一项所述抗体或抗原结合片段或权利要求15所述多特异性抗原结合分子的方法，其中，所述方法包括培养权利要求20所述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
  22. 一种制备权利要求17所述免疫效应细胞的方法，其中，所述方法包括将编码权利要求16所述的嵌合抗原受体的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求16所述的嵌合抗原受体。
  23. 一种药物组合物，其中，所述药物组合物包含权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求17所述免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述载体或根据权利要求21或22所述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
  24. 一种治疗肿瘤或癌症的方法，其中，所述方法包括向受试者施用有效量的权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求17所述免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述载体、根据权利要求20-21任一项所述方法制备获得的产品或权利要求23所述药物组合物；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
  25. 权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求17所述免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述 载体、根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述药物组合物在制备治疗肿瘤或癌症药物中的用途；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
  26. 权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求17所述免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述载体、根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述药物组合物，其中，用于治疗肿瘤或癌症；优选地，肿瘤或癌症选自实体瘤、胃癌、胃食管交界处癌、卵巢癌、输卵管癌、腹膜癌、子宫内膜癌、前列腺癌、去势抵抗前列腺癌、乳腺癌、HER2阳性乳腺癌、肉瘤、骨肉瘤、胶质母细胞瘤、肺癌、非小细胞肺癌、胆管癌、尿路上皮癌、膀胱癌、食管癌、结直肠癌、头颈癌、涎腺癌或B细胞急性淋巴细胞白血病。
  27. 一种试剂盒，其中，所述试剂盒包含权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求17所述免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述载体、根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述药物组合物。
  28. 一种检测生物学样品中HER2表达的方法，其中，所述方法包括在权利要求1～14任一项所述的抗体或抗原结合片段与HER2之间能够形成复合物的条件下，使所述生物学样品与权利要求1～14任一项所述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中HER2的存在或表达水平。
  29. 权利要求1～14任一项所述抗体或抗原结合片段在制备HER2检测试剂中的用途。
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