WO2024061224A1 - 抗her2抗体及其用途 - Google Patents

抗her2抗体及其用途 Download PDF

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Publication number
WO2024061224A1
WO2024061224A1 PCT/CN2023/119763 CN2023119763W WO2024061224A1 WO 2024061224 A1 WO2024061224 A1 WO 2024061224A1 CN 2023119763 W CN2023119763 W CN 2023119763W WO 2024061224 A1 WO2024061224 A1 WO 2024061224A1
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Prior art keywords
antibody
seq
antigen
binding fragment
her2
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PCT/CN2023/119763
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English (en)
French (fr)
Inventor
吴凡
陈连娣
缪小牛
许英达
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普米斯生物技术(珠海)有限公司
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Publication of WO2024061224A1 publication Critical patent/WO2024061224A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to the field of antibody drugs, specifically anti-HER2 antibodies and their uses.
  • HER2 also known as ErbB2. It belongs to the HER subfamily of the type I receptor tyrosine kinase family, which also includes three members: HER1 (ErbB1 or EGFR), HER3 (ErbB3) and HER4 (ErbB4). HER subfamily members can form homodimers and heterodimers, and HER2 is the strongest dimerization partner for other ErbB receptors. HER2 activation leads to receptor phosphorylation, which can trigger downstream signal cascade amplification through multiple signaling pathways such as MAPK, PI3K/AKT, JAK/STAT and PKC, and plays an important role in regulating cell proliferation, differentiation, development, adhesion and migration. effect.
  • HER2 also known as ErbB2. It belongs to the HER subfamily of the type I receptor tyrosine kinase family, which also includes three members: HER1 (ErbB1 or EGFR), HER3 (ErbB3) and HER4 (ErbB4)
  • trastuzumab is the main HER2-targeted therapeutic antibodies on the market.
  • Trastuzumab recognizes the extracellular IV domain of HER2, and Pertuzumab recognizes HER2.
  • the extracellular domain II heterodimerization site significantly improves the survival of patients.
  • trastuzumab has no therapeutic effect on many patients with tumors that also have high HER2 expression.
  • the inventor of the present application screened and obtained a series of anti-HER2 antibodies, which have high binding affinity to HER2, cross-reactivity with human and cynomolgus monkey HER2, good thermal stability, and are effective in various tumors (such as It has excellent anti-tumor effects in neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.). Accordingly, the present invention provides the following aspects.
  • the invention provides an antibody capable of specifically binding to HER2 or an antigen-binding thereof Fragments, said antibodies or antigen-binding fragments thereof comprise:
  • VH variable region
  • CDR complementarity determining regions
  • VH CDR1 which has the structure shown in GFNIKDTY (SEQ ID NO: 10);
  • VH CDR2 which has the structure shown in IYPTQGYT (SEQ ID NO: 11);
  • VH CDR3 which has the structure shown in SRWGGEGFYAMDY (SEQ ID NO: 12);
  • VL variable region
  • CDR complementarity determining regions
  • VL CDR1 having the structure shown as X 1 X 2 VQX 3 A (SEQ ID NO: 33);
  • VL CDR2 having a structure as shown in SAS (SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27);
  • VL CDR3 having the structure shown as QQHX 4 X 5 TPPT (SEQ ID NO: 34);
  • X 1 is selected from amino acid residues Q and N;
  • X 2 is selected from amino acid residues N, Y, S;
  • X 3 is selected from amino acid residues G and T;
  • X 4 is selected from amino acid residues Y, F, S;
  • X 5 is selected from amino acid residues S, M, T.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 as shown in SEQ ID NO:10
  • VH CDR2 as shown in SEQ ID NO:11
  • VH CDR3 as shown in SEQ ID NO:12
  • VL CDR1 as set forth in any of SEQ ID NOs: 18, 22, or 26, VL CDR2 as set forth in any of SEQ ID NOs: 19, 23, or 27, and, as set forth in SEQ ID NOs: 20, 24, or 28 VL CDR3 shown.
  • the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and, as set forth in SEQ ID NO: 12 The VH CDR3; and, the VL CDR1 as shown in SEQ ID NO:18, the VL CDR2 as shown in SEQ ID NO:19, and the VL CDR3 as shown in SEQ ID NO:20.
  • the antibody or antigen-binding fragment thereof comprises: such as SEQ ID NO: VH CDR1 as shown in 10, VH CDR2 as shown in SEQ ID NO:11, and VH CDR3 as shown in SEQ ID NO:12; and, VL CDR1 as shown in SEQ ID NO:22, as SEQ ID VL CDR2 shown in NO:23, and VL CDR3 shown in SEQ ID NO:24.
  • the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and, as set forth in SEQ ID NO: 12 The VH CDR3; and, the VL CDR1 as shown in SEQ ID NO:26, the VL CDR2 as shown in SEQ ID NO:27, and the VL CDR3 as shown in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO: 9 or a variant thereof, and a VL having a sequence as shown in any one of SEQ ID NOs: 17, 21 or 25 or a variant thereof;
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity; preferably, the substitution is a conservative substitution.
  • the antibody or antigen-binding fragment thereof comprises: a VH of the sequence set forth in SEQ ID NO:9, and a VL of the sequence set forth in SEQ ID NO:17.
  • the antibody or antigen-binding fragment thereof comprises: a VH of the sequence set forth in SEQ ID NO:9, and a VL of the sequence set forth in SEQ ID NO:21.
  • the antibody or antigen-binding fragment thereof comprises: a VH of the sequence set forth in SEQ ID NO:9, and a VL of the sequence set forth in SEQ ID NO:25.
  • the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgGl, IgG2, IgG3, or IgG4).
  • a human immunoglobulin eg, IgGl, IgG2, IgG3, or IgG4.
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO:29 and/or a light chain constant region as set forth in SEQ ID NO:31.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the CDR of the present invention is divided according to the IMGT division website http://aligncdr.labshare.cn/aligncdr/abrsa.php .
  • the invention provides a single-arm antibody comprising:
  • a first peptide chain wherein the first peptide chain comprises the following three heavy chain variable region (VH) complementarity determining regions (CDRs):
  • VH CDR1 which has the structure shown in GFNIKDTY (SEQ ID NO: 10);
  • VH CDR2 which has the structure shown in IYPTQGYT (SEQ ID NO: 11);
  • VH CDR3 which has the structure shown in SRWGGEGFYAMDY (SEQ ID NO: 12);
  • a second peptide chain which includes the following three light chain variable regions (VL) complementarity determining regions (CDRs):
  • VL CDR1 having the structure shown as X 1 X 2 VQX 3 A (SEQ ID NO: 33);
  • VL CDR2 having SAS (SEQ ID NO: 19, SEQ ID NO: 23 or SEQ The structure shown in ID NO:27);
  • VL CDR3 having the structure shown as QQHX 4 X 5 TPPT (SEQ ID NO: 34);
  • X 1 is selected from amino acid residues Q and N;
  • X 2 is selected from amino acid residues N, Y, S;
  • X 3 is selected from amino acid residues G and T;
  • X 4 is selected from amino acid residues Y, F, S;
  • X 5 is selected from amino acid residues S, M, T.
  • the first peptide chain includes a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and a VH CDR3 as set forth in SEQ ID NO: 12 .
  • the second peptide chain comprises a VL CDR1 as set forth in any one of SEQ ID NOs: 18, 22 or 26, a VL CDR2 as set forth in any one of SEQ ID NOs: 19, 23 or 27 , and, VL CDR3 as shown in SEQ ID NOs: 20, 24 or 28.
  • the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO: 18, a VL CDR2 as set forth in SEQ ID NO: 19, and a VL CDR3 as set forth in SEQ ID NO: 20 .
  • the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO:22, a VL CDR2 as set forth in SEQ ID NO:23, and a VL CDR3 as set forth in SEQ ID NO:24 .
  • the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO:26, a VL CDR2 as set forth in SEQ ID NO:27, and a VL CDR3 as set forth in SEQ ID NO:28 .
  • the first peptide chain comprises a heavy chain variable region (VH) of the sequence set forth in SEQ ID NO: 9 or a variant thereof, wherein the variant is consistent with the sequence from which it is derived.
  • VH heavy chain variable region
  • the first peptide chain comprises a heavy chain variable region (VH) having a sequence as shown in SEQ ID NO:9.
  • VH heavy chain variable region
  • the second peptide chain comprises a light chain variable region (VL) of a sequence set forth in any one of SEQ ID NOs: 17, 21, or 25, or a variant thereof, wherein the variant Has one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80% , at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence Sequence identity; preferably, the substitutions are conservative substitutions.
  • VL light chain variable region
  • the second peptide chain comprises a light chain variable region (VL) of a sequence set forth in any one of SEQ ID NOs: 17, 21, or 25.
  • VL light chain variable region
  • the second peptide chain further comprises a constant region derived from a human immunoglobulin.
  • the second peptide chain comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the second peptide chain comprises a light chain constant region as set forth in SEQ ID NO:31.
  • the first peptide chain further comprises a constant region derived from a human immunoglobulin.
  • the constant region derived from a human immunoglobulin is a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).
  • the heavy chain constant region has a first modification to promote dimerization of the first peptide chain and the third peptide chain.
  • the third peptide chain comprises an Fc domain monomer.
  • the Fc domain monomer is an Fc domain monomer of an IgG, such as an Fc domain monomer of IgG1, IgG2, IgG3, or IgG4.
  • the Fc domain monomer has a second modification to promote dimerization of the third peptide chain and the first peptide chain.
  • any one of the first modification and the second modification is a "knob” modification and the other is a "hole” modification to form a "knob-into-hole” modification to promote dimerization of the first peptide chain and the third peptide chain.
  • the first modification is a "knob” modification
  • the second modification is a “knob” modification to form a "knob-into-hole” modification to promote the first Dimerization of the peptide chain and the third peptide chain.
  • the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:30 and the Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO:32.
  • Node-into-acupoint technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • this method involves the introduction of ridges ("knobs") at the interface of a first polypeptide and corresponding cavities ("cavities") in the interface of a second polypeptide, such that the ridges can be placed within the cavities so that Promotes heterodimer formation and hinders homodimer formation.
  • Bumps are constructed by replacing small amino acid side chains from the first polypeptide interface with larger side chains, such as tyrosine or tryptophan.
  • Complementary cavities of the same or similar size as the ridges are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine.
  • the invention provides an isolated nucleic acid molecule encoding an antibody of the first aspect or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof; alternatively, encoding the second aspect A single-arm antibody, or its heavy chain variable region and/or light chain variable region.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a nucleotide sequence encoding said antibody or antigen-binding fragment thereof.
  • a second nucleotide sequence of a light chain or light chain variable region wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules.
  • the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a first peptide chain of the single-arm antibody of the invention or a heavy chain variable region thereof, a second peptide chain encoding the single-arm antibody or The second nucleotide sequence of its light chain variable region and the third nucleic acid sequence encoding the third peptide chain of the single-arm antibody, wherein the first nucleotide sequence, the second nucleotide sequence and The third nucleic acid sequence is present on the same or different isolated nucleic acid molecule.
  • the isolated nucleic acid molecule of the present invention includes the third nucleic acid sequence.
  • the invention provides a vector comprising the nucleic acid molecule of the third aspect.
  • the vector is a cloning vector or an expression vector.
  • the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof or A second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors.
  • the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.
  • the vector comprises a first nucleotide sequence encoding a first peptide chain of the single-arm antibody of the invention or a heavy chain variable region thereof, a second peptide chain encoding the single-arm antibody or a heavy chain variable region thereof, a second nucleotide sequence of the light chain variable region and a third nucleic acid sequence encoding the third peptide chain of the single-arm antibody, wherein the first nucleotide sequence, the second nucleotide sequence and the The third nucleic acid sequence exists on the same or different vectors.
  • the vector of the present invention includes the first nucleotide sequence.
  • a first vector, a second vector containing the second nucleotide sequence and a third vector containing the third nucleic acid sequence is included on different vectors.
  • the invention provides a host cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.) and so on.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect, which includes, allowing the antibody or antigen-binding fragment thereof or the single-arm antibody to
  • the host cell of the fifth aspect is cultured under expression conditions, and the antibody or antigen-binding fragment thereof or the single-arm antibody is recovered from the cultured host cell culture.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect, and optionally a pharmaceutically acceptable carrier and/or excipient. agent.
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • the present invention provides the antibody or antigen-binding fragment thereof of the first aspect, the single-arm antibody of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect or the host cell of the fifth aspect.
  • the present invention provides the antibody or antigen-binding fragment thereof of the first aspect, the single-arm antibody of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect or the host cell of the fifth aspect.
  • the immune cells are T cells, B cells, DC cells, macrophages, and/or NK cells.
  • the immune response is a HER2-mediated immune response.
  • the tumor is selected from solid tumors or hematological tumors (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from solid tumors or lymphomas.
  • the tumor is selected from the group consisting of neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine/cervical cancer, prostate cancer Cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.
  • the infection is selected from a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.
  • the subject is a mammal, such as a human.
  • the antibody or antigen-binding fragment thereof or the single-arm antibody is used alone or in combination with another pharmaceutically active agent.
  • the present invention provides a method for enhancing immune response in a subject, and/or preventing and/or treating tumors or infections, the method comprising: administering to a subject in need an effective An amount of the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect or the pharmaceutical composition of the seventh aspect.
  • the immune response is a HER2-mediated immune response.
  • the tumor is selected from solid tumors or hematological tumors (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from solid tumors or lymphomas.
  • the tumor is selected from the group consisting of neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine/cervical cancer, prostate cancer Cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.
  • the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections.
  • the subject is a mammal, such as a human.
  • the antibody or antigen-binding fragment thereof or single-arm antibody or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, Powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the antibody or antigen-binding fragment thereof or single-arm antibody or pharmaceutical composition of the invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection. Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared by incorporating in an appropriate solvent the requisite dose of an antibody of the invention, or an antigen-binding fragment thereof, or a single-arm antibody, and optionally other desired ingredients. (including but not limited to, pH adjuster, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) followed by filter sterilization.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • the antibody of the invention or its antigen-binding fragment, or the single-arm antibody of the invention, or the pharmaceutical composition of the invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual , globe, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that the route and/or mode of administration will vary depending on the intended purpose.
  • the antibodies or antigen-binding fragments thereof or single-arm antibodies or pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.
  • the invention provides a conjugate comprising the antibody of the first aspect or an antigen-binding fragment thereof or the single-arm antibody of the second aspect, optionally with said antibody or antigen-binding fragment thereof or said Single-arm antibody linked to a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the present invention provides a kit comprising the antibody of the first aspect or its antigen-binding fragment or the single-arm antibody of the second aspect or the conjugate of the tenth aspect.
  • the kit comprises a conjugate of the tenth aspect.
  • the kit comprises the antibody of the first aspect, or an antigen-binding fragment thereof, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.
  • the second antibody further includes a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphate acidase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
  • an enzyme e.g., horseradish peroxidase or alkaline phosphate acidase
  • chemiluminescent reagents such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent protein
  • the kit comprises the single-arm antibody of the second aspect, and a second antibody that specifically recognizes the single-arm antibody.
  • the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • an enzyme e.g., horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the invention provides a method for detecting the presence of HER2 in a sample or its level, comprising using the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect or the tenth aspect of conjugates.
  • the methods are used for therapeutic purposes, diagnostic purposes. In other embodiments, the methods are used for non-therapeutic, non-diagnostic purposes.
  • the method is an immunological assay, such as a Western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • immunological assay such as a Western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • the methods include using the conjugate of the tenth aspect.
  • the method includes using an antibody of the first aspect, or an antigen-binding fragment thereof, and the method further includes using an antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) ), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) secondary antibodies
  • a detectable label e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase)
  • chemiluminescent reagents such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent protein
  • biotin radionuclides or biotin
  • the method includes using a single-arm antibody of the second aspect, and the method further includes using a chemical carrying a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemical Secondary antibodies using luminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) are used to detect the Single-arm antibodies.
  • a detectable label such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemical Secondary antibodies using luminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) are used to detect the Single-arm antibodies.
  • an enzyme such as horseradish peroxidase or al
  • the method includes: (1) contacting the sample with an antibody of the invention or an antigen-binding fragment thereof or a single-arm antibody; (2) detecting the formation of an antigen-antibody immune complex or detecting the resulting The amount of immune complexes.
  • the formation of immune complexes indicates the presence of HER2 or HER2-expressing cells.
  • the present invention provides the use of the antibody of the first aspect or its antigen-binding fragment or the single-arm antibody of the second aspect or the conjugate of the third aspect in preparing a detection reagent for use Detect the presence or level of HER2 in the sample.
  • the detection reagent detects the presence of HER2 in the sample or the level thereof by the method for detecting the presence of HER2 in the sample or the level thereof of the twelfth aspect.
  • the sample is a sample of cells (eg, immune cells) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).
  • a subject eg, a mammal, preferably a human or a cynomolgus monkey.
  • antibody refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region.
  • the target antigen When referring to the term “antibody”, it includes not only intact antibodies but also antigen-binding fragments capable of specifically binding a target antigen, unless the context clearly indicates otherwise.
  • An “intact antibody” typically consists of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 structures domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 Definition of -917; Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, e.g. For example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgG1, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies e.g., IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.”
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody and such polypeptides, which contain sufficient antigen to confer specificity to the polypeptide At least a portion of an antibody with binding capacity.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains.”
  • “full-length heavy chain” refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the direction from the N end to the C end. It is composed of a constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
  • a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention contains two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989))
  • the term “Fab fragment” means a fragment composed of VL, VH, CL and An antibody fragment consisting of a CH1 domain
  • the term “F(ab') 2 fragment” means an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region
  • the term “Fab'fragment” means a reduction-linked F (ab') The fragment obtained after the disulfide bond of the two heavy chain fragments in the 2 fragment consists of a complete light chain and the Fd fragment of the heavy chain (composed of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "diabody” means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigens Binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • chimeric antibody refers to an antibody in which a portion of the light chain or/and heavy chain is derived from an antibody (which may originate from a specific species or belong to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains the binding activity to the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • the term “chimeric antibody” may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.
  • Fc domain or “Fc region” means a portion of the heavy chain constant region that includes CH2 and CH3.
  • the Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • Effective functions mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; effects on cell surface receptors Down-regulation of receptors (e.g., B-cell receptors); and B-cell activation, etc.
  • the Fc region can be of any antibody heavy chain constant region isotype, such as IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain may include either a native Fc region or a variant Fc region.
  • a native Fc region contains an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature, e.g., a native sequence Human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.
  • a variant Fc region includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification.
  • a variant Fc region may possess altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to the native Fc region.
  • variant Fc regions can possess modifications that promote dimerization.
  • the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence).
  • 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 as the corresponding position in the second sequence, the molecules are identical at that position.
  • Determination of percent identity between two sequences can also be accomplished using mathematical algorithms.
  • One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877.
  • Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide).
  • polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc.
  • Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art. The techniques include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H , 125I , 35S , 14C , or 32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, Histidine) amino acid.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. in some
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (eg, aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a human.
  • the subject eg, human
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent a disease e.g., tumor, infection, or autoimmune disease
  • an effective amount to treat a disease refers to an amount sufficient to cure or at least partially prevent the disease.
  • the amount of disease and its complications in patients with the disease Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • the invention provides an anti-HER2 antibody, which has high binding affinity to HER2, cross-reactivity with human and cynomolgus HER2, good thermal stability, and is effective in various tumors (such as neurocytoma, breast cancer, ovarian cancer, Gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.) Different anti-tumor effects.
  • Figure 1 shows a schematic structural diagram of the monoclonal antibody and single-arm antibody of the present invention.
  • Figure 2A and Figure 2B show the binding affinity between the monoclonal antibody of the present invention and the HER2 protein.
  • Figure 3 shows the detection of the binding of the monoclonal antibody of the present invention to human HER2 on N87 cells overexpressing HER2.
  • Figure 4 shows the detection of the monoclonal antibody of the present invention blocking HER2 signal-dependent cell proliferation on N87 cells overexpressing HER2.
  • Figure 5 shows the HER2-mediated ADCC effect of the monoclonal antibody of the present invention.
  • Figure 6 shows the charge heterogeneity analysis of the monoclonal antibody of the present invention after high temperature treatment, where OH represents the sample before high temperature treatment; HT2W, represents the sample treated with high temperature for 2 weeks; HT4W, represents the sample treated with high temperature for 4 weeks.
  • Figure 7 shows the activity verification of the monoclonal antibody of the present invention after high temperature treatment, where OH represents the sample before high temperature treatment; HT2W, represents the sample treated with high temperature for 2 weeks; HT4W, represents the sample treated with high temperature for 4 weeks.
  • Post-translational modification sites may have a negative impact on drug stability and safety, reducing drug effectiveness.
  • the present invention optimizes the Trastuzumab sequence, removes its post-translational modification site (PTM), performs random mutations on the heavy and light chain CDR regions of the antibody to construct an antibody library, and then uses yeast display technology to screen high-affinity antibodies from the antibody library. of antibodies.
  • the present invention specifically discloses the amino acid sequence encoding the antibody, and further displays relevant data of the candidate antibody.
  • Example 1 Construction and screening of monoclonal antibody affinity maturation library
  • Trastuzumab heavy chain variable region gene fragment (amino acid sequence shown as SEQ ID NO:1), light chain variable region gene fragment (amino acid sequence shown as SEQ ID NO:5) and heavy chain introducing N62Q and D110E mutations
  • Variable region gene fragment (the amino acid sequence is shown in SEQ ID NO: 9), and the light chain variable region gene fragment introducing the N36Q mutation (the amino acid sequence is shown in SEQ ID NO: 13).
  • degenerate primers were designed for the CDR2, CDR3 of VH, and the CDR3 region of VK respectively.
  • NNK was used as the mutation.
  • the base form is PCR amplified.
  • the target fragment was recovered using a PCR purification kit (purchased from QIAGEN).
  • the linearized yeast display vector and the PCR products of VH and VK were mixed and electrotransformed into Saccharomyces cerevisiae respectively. Affinity maturation libraries of heavy chain mutations and light chain mutations were constructed and the library capacity was measured.
  • Dissolve the human HER2 protein (purchased from AcroBiosystems) in an appropriate volume of double-distilled water. According to the product instructions of the biotin labeling kit (purchased from Thermo), dissolve the biotin and mix it with the protein solution, and incubate at 4°C for 2 hours. Use a desalting column (purchased from Thermo) to remove excess biotin. The desalting column pretreatment and sample collection operations are performed according to the product instructions.
  • the antibody library constructed in Example 1.1 was inoculated into SD-CAA amplification medium (1L SD-CAA amplification medium contained 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4 ⁇ 12H2O, 7.44g NaH2PO4 and 2% glucose ), incubate overnight at 30°C, 225rpm. Take an appropriate amount of yeast cells, centrifuge at 3000 rpm ⁇ 5 min (the following centrifugation operations are the same) to remove the culture medium, resuspend the yeast cells in SD-CAA induction medium, and induce overnight. Determine the library concentration after induction, take an appropriate amount of yeast cells, and centrifuge to remove the culture medium. Resuspend the yeast cells in 50ml PBS and centrifuge to remove the supernatant. Resuspend yeast cells in 10ml PBS.
  • Biotin-labeled human HER2 protein (final concentration 100 nM), incubate at room temperature for 30 minutes, collect yeast cells by centrifugation, and wash the yeast three times with 50 ml PBS. Resuspend the yeast cells in 5 ml of washing solution, add 200 ⁇ l of SA magnetic beads (purchased from Miltenyi), and incubate upside down for 10 min. The yeast and magnetic bead mixture was washed three times with PBS, and the mixture was added to an LS purification column (purchased from Miltenyi). Place the LS purification column on a magnetic stand and wash with PBS to remove non-specifically bound yeast cells. Remove the purification column from the magnetic stand and add PBS to elute the yeast. The eluted yeast was centrifuged and transferred to SD-CAA amplification medium for amplification.
  • MACS-enriched yeast cells were inoculated into SD-CAA expansion medium. Incubate in shake flask at 30°C, 225rpm overnight. Resuspend yeast cells in SD-CAA induction medium and induce overnight. Add anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled HER2 antigen, and incubate for 10 minutes. Add PBS to wash the yeast 3 times, add goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin APC conjugate fluorescent antibody (purchased from Invitrogen), and incubate for 15 minutes. Add PBS to resuspend the cells, and use the BD FACS AriaIII instrument to sort the yeast to obtain yeast with higher binding ability to the HER2 antigen.
  • the yeast liquid with high binding ability to human HER2 antigen obtained through MACS and FACS enrichment is spread on the SD-CAA solid culture plate, and then single clones are picked in the SD-CAA amplification medium for 30 C, 225 rpm overnight, treat the amplified single clone with 0.1% SDS, centrifuge and use the supernatant as a template for PCR amplification, and send the PCR product to sequencing to obtain the gene sequence.
  • the heavy chain variable region gene sequence to the human IgG1 constant region (the amino acid sequence is shown in SEQ ID NO: 29), and use homologous recombinase (purchased from Vazyme) to construct an EcoR I/Not I double enzyme digestion linearized In the pCDNA3.1 vector; the light chain variable region gene sequence is connected to the human Kappa constant region (the amino acid sequence is shown in SEQ ID NO: 31), and constructed into the EcoR I/Xhol I double enzyme digested linearized pCDNA3.1 vector , the process is in accordance with the product manual.
  • the homologous recombination products were transformed into Top10 competent cells, spread on ampicillin-resistant plates, cultured at 37°C overnight, single clones were picked for sequencing, and plasmids were extracted.
  • the heavy chain variable region gene sequence was connected to the human IgG1 constant region containing Knob mutation (the amino acid sequence is shown in SEQ ID NO: 30), and homologous recombinase (purchased from Vazyme) was used.
  • the light chain variable region gene sequence is connected to the human Kappa light chain constant region (the amino acid sequence is shown in SEQ ID NO: 31), and constructed into EcoR In the pCDNA3.1 vector linearized by I/Xhol I double digestion, the human Fc region encoding the human Fc region containing the Hole mutation (the amino acid sequence is shown in SEQ ID NO: 32) was constructed into pCDNA3 linearized by EcoRI/XhoI double digestion. 1 in the carrier.
  • the process follows the product manual. The homologous recombination product was transferred into Top10 competent cells, coated on ampicillin-resistant plates, and cultured at 37°C overnight. Single clones were picked for sequencing, and plasmids were extracted. Cell transfection and protein purification were performed according to Example 2.2.
  • HPLC HPLC to detect protein purity.
  • the HPLC method is as follows, mobile phase: 150mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m).
  • the affinity-purified antibody molecules of the present invention have good purity and meet the requirements of downstream process development.
  • DSC Different scanning calorimetry
  • the sample was concentrated and diluted to 1 mg/ml with PBS; the 5000 ⁇ fluorescent reagent Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100 ⁇ fluorescent reagent Sypro Orange.
  • ForteBio affinity assay was performed according to existing methods (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput method. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in the analysis buffer for 30 min, then detected online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM human or cynomolgus monkey HER2 antigen (purchased from AcroBiosystems) for 5 min, and then the sensor was transferred to the analysis buffer for dissociation for 5 min. Finally, a 1:1 binding model was used for kinetic analysis.
  • the affinity of the Anti-HER2 antibody optimized in the present invention is equivalent to that of Trastuzumab before optimization.
  • the affinity of the Trastuzumab PTM removal single-arm antibody is significantly weaker than that of the Trastuzumab single-arm antibody, and the Anti-HER2 single-arm antibody of the present invention after PTM modification and affinity optimization has an affinity for binding to human HER2 antigen that is no less than that of the Trastuzumab single-arm antibody.
  • expanded cultured N87 cells self-expressing HER2 cells were treated with 0.25% EDTA Digest trypsin, wash once with culture medium, adjust the cell density to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, centrifuge and set aside. Add the serially diluted antibody at 100 ⁇ l/well to the above-mentioned 96-well flow cytometry plate with cells, and incubate at 4°C for 60 minutes. After washing twice with PBS, 100 ⁇ l/well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000 times with 2% BSA solution was added, and incubated at 4°C for 60 min. Wash twice with PBS, and finally add 100 ⁇ l/well of PBS to resuspend the cells. Detect the cells on a CytoFlex (Beckman) flow cytometer and calculate the corresponding mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • the binding activity of the optimized Anti-HER2 antibody in the present invention to HER2 expressed on human gastric cancer cell N87 is comparable to that of Trastuzumab.
  • the cell-binding activity of the Trastuzumab PTM removal single-arm antibody is significantly weaker than that of the Trastuzumab single-arm antibody.
  • the affinity-optimized Anti-HER2 single-arm antibody binds to the HER2 antigen on N87 with an affinity that is no less than that of the Trastuzumab single-arm antibody, and is very similar to the single-arm antibody. quite.
  • the expanded cultured N87 (self-expressing HER2) cells were digested with 0.25% EDTA trypsin, washed once with culture medium, and then adjusted to a cell density of 5 ⁇ 10 4 cells/ml. 80 ⁇ l/well was added to a 96-well plate for later use. Add 80 ⁇ l/well of the serially diluted antibody to the above-mentioned 96-well plate with cells, and place it in a cell culture incubator for incubation for 3-5 days. Use last After color development with the Luminescent Cell Viability Assay (Promega, G7572) kit, the chemiluminescence signal was collected using a microplate reader.
  • the optimized Anti-HER2 monoclonal antibodies in the present invention can significantly inhibit the proliferation of N87 cells, and the cell proliferation inhibitory effect is equivalent to that of Trastuzumab monoclonal antibody.
  • the expanded cultured N87 (self-expressing HER2) cells were mixed and inoculated into 3 In a 96-well cell culture white bottom plate, the HER2 monoclonal antibody was then added to the 96-well plate after gradient dilution, mixed, and placed in a cell culture incubator for 6 hours.
  • the Bio-Glo luciferase assay system Promega, G7940
  • the optimized Anti-HER2 monoclonal antibodies in the present invention can mediate ADCC through HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells.
  • the ADCC effect of Anti-HER2(NO3-46) antibody is comparable to that of Trastuzumab antibody.
  • the protein sample was diluted to 1 mg/ml with diluent, divided into vials, and then placed in a 4°C refrigerator and a 40°C incubator for incubation for 2 weeks and 4 weeks respectively. After the samples were treated at high temperature for 2 weeks and 4 weeks, antibody characterization analysis and activity verification were performed, such as charge heterogeneity analysis, PTM analysis and activity verification, etc.
  • CEX-HPLC Cation exchange chromatography
  • N/A means not detected
  • ForteBio affinity assay was performed according to existing methods (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput method. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in the analysis buffer for 30 min, then detected online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM human HER2 antigen (purchased from AcroBiosystems) for 5 minutes, and then the sensor was transferred to the analysis buffer for dissociation for 5 minutes. Finally, a 1:1 binding model was used for kinetic analysis.

Abstract

抗HER2抗体及其用途，具体提供了系列抗HER2抗体，其与HER2的结合亲和力高，具备与人、食蟹猴HER2的交叉反应活性，热稳定性良好，在多种肿瘤(如神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌等)中具有优异的抗肿瘤效果。

Description

抗HER2抗体及其用途 技术领域
本发明涉及抗体药物领域，具体涉及抗HER2抗体及其用途。
背景技术
HER2,又名ErbB2。它属于I型受体酪氨酸激酶家族中的HER亚家族，亚家族中还包括HER1(ErbB1或EGFR)、HER3(ErbB3)和HER4(ErbB4)等3个成员。HER亚家族成员可形成同二聚体和异二聚体，HER2是其它ErbB受体的最强二聚配偶体。HER2活化导致受体磷酸化，可通过MAPK、PI3K/AKT、JAK/STAT和PKC等多条信号通路触发下游的信号级联放大，对细胞的增殖、分化、发育、黏附及迁移起重要的调节作用。研究人员发现HER2的高表达与很多肿瘤有关，如：神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌等等。
目前，曲妥珠单抗(Trastuzumab)和帕妥珠单抗(Pertuzumab)是市售的主要HER2靶向治疗抗体，曲妥珠单抗识别HER2胞外IV结构域，帕妥珠单抗识别HER2细胞外结构域II异源二聚化位点，显著的改善了患者的生存期。然而，研究人员也发现，曲妥珠单抗(Trastuzumab)对许多同样是HER2高表达的肿瘤病人没有治疗效果。
因此，本领域仍然存在开发新的抗HER2抗体需求。
发明内容
本申请的发明人经过大量的研究，筛选获得了系列抗HER2抗体，其与HER2的结合亲和力高，具备与人、食蟹猴HER2的交叉反应活性，热稳定性良好，在多种肿瘤(如神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌等)中具有优异的抗肿瘤效果。由此，本发明提供了以下方面。
抗体或其抗原结合片段
在第一方面，本发明提供了能够特异性结合HER2的抗体或其抗原结合 片段，所述抗体或其抗原结合片段包含：
(1)下述3个重链可变区(VH)互补决定区(CDR)：
(a)VH CDR1，其具有如GFNIKDTY(SEQ ID NO:10)所示的结构；
(b)VH CDR2，其具有如IYPTQGYT(SEQ ID NO:11)所示的结构；
(c)VH CDR3，其具有如SRWGGEGFYAMDY(SEQ ID NO:12)所示的结构；
和/或，
(2)下述3个轻链可变区(VL)互补决定区(CDR)：
(d)VL CDR1，其具有如X1X2VQX3A(SEQ ID NO:33)所示的结构；
(e)VL CDR2，其具有如SAS(SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27)所示的结构；
(f)VL CDR3，其具有如QQHX4X5TPPT(SEQ ID NO:34)所示的结构；
其中：
X1选自氨基酸残基Q、N；
X2选自氨基酸残基N、Y、S；
X3选自氨基酸残基G、T；
X4选自氨基酸残基Y、F、S；
X5选自氨基酸残基S、M、T。
在一些实施方案中，所述抗体或其抗原结合片段包含：
如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，
如SEQ ID NOs:18、22或26任一项所示的VL CDR1，如SEQ ID NOs:19、23或27任一项所示的VL CDR2，以及，如SEQ ID NOs:20、24或28所示的VL CDR3。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:18所示的VL CDR1，如SEQ ID NO:19所示的VL CDR2，以及，如SEQ ID NO:20所示的VL CDR3。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO: 10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:22所示的VL CDR1，如SEQ ID NO:23所示的VL CDR2，以及，如SEQ ID NO:24所示的VL CDR3。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:26所示的VL CDR1，如SEQ ID NO:27所示的VL CDR2，以及，如SEQ ID NO:28所示的VL CDR3。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:9所示的序列或其变体的VH，和，如SEQ ID NOs:17、21或25任一项所示的序列或其变体的VL；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；优选地，所述的置换是保守置换。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:17所示的序列的VL。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:21所示的序列的VL。
在一些实施方案中，所述抗体或其抗原结合片段包含：如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:25所示的序列的VL。
在一些实施方案中，所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区。
在一些实施方案中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区。
在一些实施方案中，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在一些实施方案中，所述抗体或其抗原结合片段进一步包含如SEQ ID NO:29所示的重链恒定区和/或如SEQ ID NO:31所示的轻链恒定区。
在一些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
本发明的CDR是按照IMGT划分网站http://aligncdr.labshare.cn/aligncdr/abrsa.php划分得到的。
单臂抗体
在第二方面，本发明提供了单臂抗体，其包含：
(1)第一肽链，所述第一肽链包含下述3个重链可变区(VH)互补决定区(CDR)：
(a)VH CDR1，其具有如GFNIKDTY(SEQ ID NO:10)所示的结构；
(b)VH CDR2，其具有如IYPTQGYT(SEQ ID NO:11)所示的结构；
(c)VH CDR3，其具有如SRWGGEGFYAMDY(SEQ ID NO:12)所示的结构；
(2)第二肽链，所述第二肽链包含下述3个轻链可变区(VL)互补决定区(CDR)：
(d)VL CDR1，其具有如X1X2VQX3A(SEQ ID NO:33)所示的结构；
(e)VL CDR2，其具有如SAS(SEQ ID NO:19、SEQ ID NO:23或SEQ  ID NO:27)所示的结构；
(f)VL CDR3，其具有如QQHX4X5TPPT(SEQ ID NO:34)所示的结构；
和，
(3)第三肽链，所述第三肽链能够和所述第一肽链形成二聚体；
其中：
X1选自氨基酸残基Q、N；
X2选自氨基酸残基N、Y、S；
X3选自氨基酸残基G、T；
X4选自氨基酸残基Y、F、S；
X5选自氨基酸残基S、M、T。
在一些实施方案中，所述第一肽链包含如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3。
在一些实施方案中，所述第二肽链包含如SEQ ID NOs:18、22或26任一项所示的VL CDR1，如SEQ ID NOs:19、23或27任一项所示的VL CDR2，以及，如SEQ ID NOs:20、24或28所示的VL CDR3。
在一些实施方案中，所述第二肽链包含如SEQ ID NO:18所示的VL CDR1，如SEQ ID NO:19所示的VL CDR2，以及，如SEQ ID NO:20所示的VL CDR3。
在一些实施方案中，所述第二肽链包含如SEQ ID NO:22所示的VL CDR1，如SEQ ID NO:23所示的VL CDR2，以及，如SEQ ID NO:24所示的VL CDR3。
在一些实施方案中，所述第二肽链包含如SEQ ID NO:26所示的VL CDR1，如SEQ ID NO:27所示的VL CDR2，以及，如SEQ ID NO:28所示的VL CDR3。
在一些实施方案中，所述第一肽链包含如SEQ ID NO:9所示的序列或其变体的重链可变区(VH)，其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少 91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；优选地，所述的置换是保守置换。
在一些实施方案中，所述第一肽链包含如SEQ ID NO:9所示的序列的重链可变区(VH)。
在一些实施方案中，所述第二肽链包含如SEQ ID NOs:17、21或25任一项所示的序列或其变体的轻链可变区(VL)，其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；优选地，所述的置换是保守置换。
在一些实施方案中，所述第二肽链包含如SEQ ID NOs:17、21或25任一项所示的序列的轻链可变区(VL)。
在一些实施方案中，所述第二肽链进一步包含来源于人免疫球蛋白的恒定区。
在一些实施方案中，所述第二肽链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在一些实施方案中，所述第二肽链包含如SEQ ID NO:31所示的轻链恒定区。
在一些实施方案中，所述第一肽链进一步包含来源于人免疫球蛋白的恒定区。在一些实施方案中，所述来源于人免疫球蛋白的恒定区为来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区。在一些实施方案中，所述重链恒定区具有第一修饰，以促进所述第一肽链和所述第三肽链的二聚化。
在一些实施方案中，所述第三肽链包含Fc结构域单体。在一些实施方案中，所述Fc结构域单体是IgG的Fc结构域单体，例如IgG1、IgG2、IgG3或IgG4的Fc结构域单体。在一些实施方案中，所述Fc结构域单体具有第二修饰，以促进所述第三肽链和所述第一肽链的二聚化。
在一些实施方案中，所述第一修饰和所述第二修饰中，任意一个为“节”修饰，另一个为“穴”修饰，以形成节-入-穴(knob-into-hole)”修饰，促进所述第一肽链和所述第三肽链的二聚化。
在一些实施方案中，所述第一修饰为“节”修饰，所述第二修饰为“穴”修饰，以形成节-入-穴(knob-into-hole)”修饰，促进所述第一肽链和所述第三肽链的二聚化。
在一些实施方案中，所述重链恒定区包含如SEQ ID NO:30所示的氨基酸序列，所述Fc结构域单体包含如SEQ ID NO:32所示的氨基酸序列。
节-入-穴技术记载于例如US 5,731,168；US 7,695,936；Ridgway等，Prot Eng 9，617-621(1996)和Carter，J Immunol Meth 248，7-15(2001)。一般地，该方法牵涉在第一多肽的界面处引入隆起(“节”)并在第二多肽的界面中引入相应的空腔(“穴”)，使得隆起可以置于空腔中从而促进异二聚体形成并阻碍同二聚体形成。通过将来自第一多肽界面的小氨基酸侧链用更大的侧链(例如酪氨酸或色氨酸)替换来构建隆起。在第二多肽的界面中创建具有与隆起相同或相似大小的互补性空腔，其通过将大氨基酸侧链用更小的氨基酸侧链(例如丙氨酸或苏氨酸)替换进行。
分离的核酸分子
在第三方面，本发明提供了分离的核酸分子，其编码第一方面的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；或者，其编码第二方面的单臂抗体，或其重链可变区和/或轻链可变区。
在一些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的分离的核酸分子上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的分离的核酸分子上时，本发明所述的分离的核酸分子包含含有所述第一核苷酸序列的第一核酸分子以及含有所述第二核苷酸序列的第二核酸分子。
在一些实施方案中，所述分离的核酸分子包含编码本发明单臂抗体的第一肽链或其重链可变区的第一核苷酸序列、编码所述单臂抗体的第二肽链或 其轻链可变区的第二核苷酸序列和编码所述单臂抗体的第三肽链的第三核酸序列，其中所述第一核苷酸序列、所述第二核苷酸序列和所述第三核酸序列存在于相同或不同的分离的核酸分子上。当所述第一核苷酸序列、所述第二核苷酸序列和所述第三核酸序列存在于不同的分离的核酸分子上时，本发明所述的分离的核酸分子包含含有所述第一核苷酸序列的第一核酸分子、含有所述第二核苷酸序列的第二核酸分子和含有所述第三核酸序列的第三核酸分子。
载体
在第四方面，本发明提供了载体，其包含第三方面的核酸分子。在一些实施方案中，所述载体为克隆载体或表达载体。
在一些实施方案中，所述载体包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的载体上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的载体上时，本发明所述的载体包含含有所述第一核苷酸序列的第一载体以及含有所述第二核苷酸序列的第二载体。
在一些实施方案中，所述载体包含编码本发明的单臂抗体的第一肽链或其重链可变区的第一核苷酸序列、编码所述单臂抗体的第二肽链或其轻链可变区的第二核苷酸序列和编码所述单臂抗体的第三肽链的第三核酸序列，其中所述第一核苷酸序列、所述第二核苷酸序列和所述第三核酸序列存在于相同或不同的载体上。当所述第一核苷酸序列、所述第二核苷酸序列和所述第三核酸序列存在于不同的载体上时，本发明所述的载体包含含有所述第一核苷酸序列的第一载体、含有所述第二核苷酸序列的第二载体和含有所述第三核酸序列的第三载体。
宿主细胞
在第五方面，本发明提供了宿主细胞，其包含如第三方面的核酸分子或第四方面的载体。此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)等等。
制备方法
在第六方面，本发明提供了制备第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体的方法，其包括，在允许所述抗体或其抗原结合片段或者所述单臂抗体表达的条件下，培养第五方面的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段或者所述单臂抗体。
治疗用途
在第七方面，本发明提供了药物组合物，其包含如第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体，以及任选的药学上可接受的载体和/或赋形剂。
在某些示例性实施方案中，所述药学上可接受的载体和/或赋形剂包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在第八方面，本发明提供了第一方面的抗体或其抗原结合片段、第二方面的单臂抗体、第三方面的分离的核酸分子、第四方面的载体或第五方面的宿主细胞用于制备药物的用途，所述药物用于在受试者中激活HER2，提高免疫细胞活性，增强免疫应答，和/或预防和/或治疗肿瘤或者感染。
在一些实施方案中，所述免疫细胞是T细胞，B细胞，DC细胞，巨噬细胞，和/或，NK细胞。
在一些实施方案中，所述免疫应答是HER2介导的免疫应答。
在一些实施方案中，所述肿瘤选自实体肿瘤或血液肿瘤(例如，白血病、淋巴瘤、骨髓瘤)。在某些实施方案中，所述肿瘤选自实体肿瘤或淋巴瘤。
在一些实施方案中，所述肿瘤选自神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌、结直肠癌、结肠癌、子宫/宫颈癌、前列腺癌、睾丸癌、食管癌、胃肠癌、头颈癌、生殖细胞癌、骨癌、肝癌、甲状腺癌、皮肤癌、中枢神经系统的肿瘤、淋巴瘤、白血病、骨髓瘤、肉瘤、黑色素瘤。
在一些实施方案中，所述感染选自病毒感染、细菌感染、真菌感染和寄生虫感染。
在一些实施方案中，所述受试者为哺乳动物，例如人。
在一些实施方案中，所述抗体或其抗原结合片段或者所述单臂抗体单独使用，或与另外的药学活性剂联合使用。
在第九方面，本发明提供了一种在受试者中增强免疫应答，和/或，预防和/或治疗肿瘤或感染的方法，所述方法包括：给有此需要的受试者施用有效量的第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体或者第七方面的药物组合物。
在一些实施方案中，所述免疫应答是HER2介导的免疫应答。
在一些实施方案中，所述肿瘤选自实体肿瘤或血液肿瘤(例如，白血病、淋巴瘤、骨髓瘤)。在某些实施方案中，所述肿瘤选自实体肿瘤或淋巴瘤。
在一些实施方案中，所述肿瘤选自神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌、结直肠癌、结肠癌、子宫/宫颈癌、前列腺癌、睾丸癌、食管癌、胃肠癌、头颈癌、生殖细胞癌、骨癌、肝癌、甲状腺癌、皮肤癌、中枢神经系统的肿瘤、淋巴瘤、白血病、骨髓瘤、肉瘤、黑色素瘤。
在一些实施方案中，所述感染选自病毒感染、细菌感染、真菌感染和寄生虫感染。
在一些实施方案中，所述受试者为哺乳动物，例如人。
本发明的抗体或其抗原结合片段或者单臂抗体或者药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的抗体或其抗原结合片段或者单臂抗体或者药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段或者单臂抗体，以及任选地，同时掺入其他期望的成分(包括但不限于， pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明的抗体或其抗原结合片段、或者本发明的单臂抗体、或者本发明的药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。本领域技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明的抗体或其抗原结合片段或者单臂抗体或者药物组合物通过静脉注射或推注给予。
检测用途
在第十方面，本发明提供了缀合物，其包含第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体，以及任选的与所述抗体或其抗原结合片段或者所述单臂抗体连接的可检测的标记。
在一些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
在第十一方面，本发明提供了试剂盒，其包含第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体或者第十方面的缀合物。
在一些实施方案中，所述试剂盒包含第十方面的缀合物。
在一些实施方案中，所述试剂盒包含第一方面的抗体或其抗原结合片段，以及特异性识别所述抗体或其抗原结合片段的第二抗体。在某些实施方案中，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷 酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
在一些实施方案中，所述试剂盒包含第二方面的单臂抗体，以及特异性识别所述单臂抗体的第二抗体。在某些实施方案中，所述第二抗体还包含可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
在第十二方面，本发明提供了用于检测HER2在样品中的存在或其水平的方法，其包括使用第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体或者第十方面的缀合物。在某些实施方案中，所述方法用于治疗目的，诊断目的。在另一些实施方案中，所述方法用于非治疗非诊断目的。
在一些实施方案中，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在一些实施方案中，所述方法包括使用第十方面的缀合物。
在一些实施方案中，所述方法包括使用第一方面的抗体或其抗原结合片段，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述抗体或其抗原结合片段。
在一些实施方案中，所述方法包括使用第二方面的单臂抗体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述单臂抗体。
在某些实施方案中，所述方法包括：(1)将所述样品与本发明的抗体或其抗原结合片段或者单臂抗体接触；(2)检测抗原-抗体免疫复合物的形成或检测所述免疫复合物的量。所述免疫复合物的形成表明存在HER2或表达HER2的细胞。
在第十三方面，本发明提供了第一方面的抗体或其抗原结合片段或者第二方面的单臂抗体或者第三方面的缀合物在制备检测试剂中的用途，所述检测试剂用于检测HER2在样品中的存在或其水平。
在一些实施方案中，所述检测试剂通过第十二方面的用于检测HER2在样品中的存在或其水平的方法来检测HER2在样品中的存在或其水平。
在一些实施方案中，所述样品为来自受试者(例如哺乳动物，优选人或食蟹猴)的细胞样品(例如，免疫细胞)。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
本文所用的术语“抗体”是指能够特异性结合靶抗原的源自免疫球蛋白的分子，所述源自免疫球蛋白的分子通过位于其可变区中的至少一个抗原结合位点来结合所述靶抗原。当提及术语“抗体”时，除非上下文明确指出，其不仅包括完整抗体，而且包括能够特异性结合靶抗原的抗原结合片段。“完整抗体”典型地由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构 域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例 如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、probody和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”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。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”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。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和 CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原 结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体，其中抗体的重链和轻链可变区来自第一抗体，而抗体的重链和轻链恒定区来自第二抗体。
如本文中所使用的，术语“Fc结构域”或“Fc区域”意指，包含CH2和CH3的重链恒定区的一部分。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。由Fc区介导的“效应子功能”包括Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。Fc区可为任何抗体重链恒定区同型，例如IgG1、IgG2、IgG3或IgG4。
Fc结构域既可以包括天然Fc区，也可以包括变异Fc区。天然Fc区包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列，例如天然序列 人类Fc区包括天然序列人类IgG1Fc区(非A和A同种异型)；天然序列人类IgG2Fc区；天然序列人类IgG3Fc区；及天然序列人类IgG4Fc区，以及其天然存在的变异体。变异Fc区包含因至少一个氨基酸修饰而与天然序列Fc区的氨基酸序列不同的氨基酸序列。在一些实施方案中，变异Fc区可具备相比于天然Fc区改变的效应子功能(例如Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。在一些实施方案中，变异Fc区可具备促进二聚化的修饰。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较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被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990,Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人,1990,J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所 述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物、鲁米诺及其衍生物、钌衍生物如三联吡啶钌)、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、 组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些 示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中所使用的，术语“受试者”是指哺乳动物，例如人。在某些实施方案中，所述受试者(例如人)患有肿瘤、感染或自身免疫性疾病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤、感染或自身免疫性疾病)有效量是指，足以预防，阻止，或延迟所述疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明提供了抗HER2抗体，其与HER2的结合亲和力高，具备与人、食蟹猴HER2的交叉反应活性，热稳定性良好，在多种肿瘤(如神经细胞瘤、乳腺癌、卵巢癌、胃癌、肺癌、肾癌、肠癌、胰腺癌、膀胱癌等)中具有优 异的抗肿瘤效果。
附图说明
图1显示本发明所述单克隆抗体和单臂抗体结构示意图。
图2A、图2B显示本发明所述的单克隆抗体与HER2蛋白的结合亲和力。
图3显示在过表达HER2的N87细胞上检测本发明所述的单克隆抗体与人HER2的结合情况。
图4显示在过表达HER2的N87细胞上检测本发明所述单克隆抗体阻断HER2信号依赖的细胞增殖。
图5显示本发明所述单克隆抗体通过HER2介导的ADCC作用。
图6显示本发明所述单克隆抗体在高温处理后的电荷异质性分析，其中0H表示高温处理前的样品；HT2W，表示高温处理2周的样品；HT4W，表示高温处理4周的样品。
图7显示本发明所述的单克隆抗体在高温处理后的活性验证，其中0H表示高温处理前的样品；HT2W，表示高温处理2周的样品；HT4W，表示高温处理4周的样品。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
序列信息
翻译后修饰位点(PTM)可能对药物的稳定性和安全性有负面影响，降低药物的效果。本发明对Trastuzumab序列进行了优化，去除了其翻译后修饰位点(PTM)，并对抗体的重轻链CDR区域做随机突变构建抗体文库，然后通过酵母展示技术从抗体库中筛选出高亲和力的抗体。本发明具体公开了编码该抗体的氨基酸序列，并进一步展示了候选抗体的相关数据。
本发明涉及的序列的描述提供于下表1-1和表1-2中。
表1-1.本发明的序列可变区和CDR总结
表1-2.本发明的序列恒定区和其他序列总结



具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：单克隆抗体亲和力成熟文库的构建及筛选
1.1.亲和成熟文库构建
合成Trastuzumab重链可变区基因片段(氨基酸序列如SEQ ID NO:1所示)、轻链可变区基因片段(氨基酸序列如SEQ ID NO:5所示)以及引入N62Q和D110E突变的重链可变区基因片段(氨基酸序列如SEQ ID NO:9所示)、引入N36Q突变的轻链可变区基因片段(氨基酸序列如SEQ ID NO:13所示)。以去除PTM的VH和VK为模板，分别对VH的CDR2、CDR3，VK的CDR3区域进行简并引物设计，为保证每个氨基酸都能突变成20种氨基酸的任何一种，以NNK作为突变的碱基形式进行PCR扩增。利用PCR纯化试剂盒(购自QIAGEN)回收目的片段。将线性化的酵母展示载体和VH及VK的PCR产物混合后分别电转化入酿酒酵母中，分别构建重链突变和轻链突变的亲和力成熟文库并测定库容。
1.2.抗HER2抗体的筛选
1.2.1 HER2蛋白的生物素化标记
取适量体积的双蒸水溶解人HER2蛋白(购自AcroBiosystems)，按照生物素标记试剂盒(购自Thermo)产品说明书，将生物素溶解后与蛋白溶液混合，于4℃孵育2小时。用脱盐柱(购自Thermo)去除多余的生物素，脱盐柱预处理及样品收集操作均参考产品说明书步骤进行。
1.2.2 MACS富集能与人HER2特异性结合的酵母
将实施例1.1中构建的抗体文库接种于SD-CAA扩增培养基(1L SD-CAA扩增培养基中含6.7g YNB、5g酪氨基酸、13.62g Na2HPO4·12H2O、7.44g NaH2PO4和2％葡萄糖)中，30℃，225rpm培养过夜。取适量酵母细胞，3000rpm×5min离心(以下离心操作均同此)去除培养基，用SD-CAA诱导培养基重悬酵母细胞，诱导过夜。测定诱导后的文库浓度，取适量酵母细胞，离心去除培养基。用50ml PBS重悬酵母细胞，离心去除上清。用10ml PBS重悬酵母细胞。
加入生物素标记的人HER2蛋白(终浓度100nM)，室温孵育30min，离心收集酵母细胞，并用50ml PBS洗涤酵母3遍。用5ml清洗液重悬酵母细胞，并加入200μl SA磁珠(购自美天旎)，颠倒孵育10min。用PBS洗涤酵母和磁珠混合物3遍，将混合物加入LS纯化柱(购自美天旎)中。将LS纯化柱放在磁力架上，用PBS洗涤去除非特异性结合的酵母细胞。将纯化柱从磁力架上取出，加入PBS洗脱酵母。洗脱下来的酵母离心后转入SD-CAA扩增培养基中进行扩增。
1.2.3流式细胞分选获得高亲和力酵母细胞
将经过MACS富集的酵母细胞接种于SD-CAA扩增培养基中。30℃，225rpm摇瓶培养过夜。用SD-CAA诱导培养基重悬酵母细胞，诱导过夜。加入抗-c-Myc鼠源抗体(购自Thermo)和100nM生物素标记的HER2抗原，孵育10min。加入PBS清洗酵母3遍，加入羊抗小鼠IgG(H+L)Alexa Fluor Plus 488荧光抗体(购自Invitrogen)和链霉亲和素APC结合物荧光抗体(购自Invitrogen)，孵育15min。加入PBS重悬细胞，使用BD FACS AriaIII仪器进行分选获得可与HER2抗原有较高结合能力的酵母。
1.2.4 HER2抗体候选分子抗体基因的调取
通过MACS和FACS富集得到的能与人HER2抗原有较高结合能力的酵母菌液，涂布于SD-CAA的固体培养板上，然后挑取单克隆在SD-CAA扩增培养基中30℃，225rpm培养过夜，用0.1％的SDS处理扩增后的单克隆，离心并以上清作为模板进行PCR扩增，将PCR产物送测序以获得基因序列。
实施例2：单克隆抗体的构建及表达纯化
2.1.单克隆抗体基因构建入pCDNA3.1表达载体
将重链可变区基因序列和人IgG1恒定区(氨基酸序列如SEQ ID NO:29所示)相连，利用同源重组酶(购自Vazyme)构建到EcoR I/Not I双酶切线性化的pCDNA3.1载体中；轻链可变区基因序列和人Kappa恒定区(氨基酸序列如SEQ ID NO:31所示)相连，构建到EcoR I/Xhol I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序，并抽提质粒。
2.2.细胞转染及蛋白纯化
采用HEK293细胞进行转染表达，转染前一天将细胞密度调整至2.5×106细胞/ml，次日转染时稀释到3.0×106细胞/ml使用，以MEM培养基作为转染缓冲液，按PEI：质粒＝3:1比例加入PEI，将抽提的重链、轻链两种质粒共转入HEK293细胞中，细胞培养5天后收集上清利用Protein A填料柱纯化目的蛋白。向填料柱中加10倍柱体积的PBS平衡柱子，将上述细胞上清加入到重力柱中，重力流穿，上样结束后，用20倍柱体积的PBS洗去不结合的杂蛋白，待没有液体流出后，将柱子放置于提前加好中和缓冲液(1M Tris,pH8.54)的收集管上，用3-5倍柱体积的洗脱缓冲液(0.1M柠檬酸钠,pH3.2)洗脱获得目的蛋白。
实施例3：Anti-HER2单臂抗体的构建及表达纯化
为进一步确认单价抗体的亲和力情况，将重链可变区基因序列和含有Knob突变的人IgG1恒定区(氨基酸序列如SEQ ID NO:30所示)相连，利用同源重组酶(购自Vazyme)构建到EcoR I/Not I双酶切线性化的pCDNA3.1载体中；轻链可变区基因序列和人Kappa轻链恒定区(氨基酸序列如SEQ ID NO:31所示)相连，构建到EcoR I/Xhol I双酶切线性化的pCDNA3.1载体中，将编码含有Hole突变的人Fc区域(氨基酸序列如SEQ ID NO:32所示)构建到EcoRⅠ/XhoⅠ双酶切线性化的pCDNA3.1载体中。 流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序，并抽提质粒，并参照实施例2.2进行细胞转染及蛋白纯化。
实施例4：Anti-HER2抗体的纯度测定
本研究利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na2HPO4·12H2O,pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20min，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm,3μm)。
结果如表2所示，亲和纯化后的本发明的抗体分子具有较好的纯度，满足下游工艺开发的要求。
表2.Anti-HER2抗体的纯度检测结果
实施例5：Anti-HER2单克隆抗体的热稳定性
利用DSC(Differential scanning calorimetry,差示扫描量热法)检测不同抗体的热稳定性。将样品浓缩后用PBS稀释到1mg/ml；将5000×荧光显色剂Cypro Orange(购于Bio-Rad)用超纯水稀释50倍得到100×荧光显色剂Sypro Orange。取50μl 1mg/ml的样品加入10μl 100×荧光显色剂Sypro  Orange、40μl超纯水，混匀后，取30μl加入到96孔PCR板中，每个样品做3个复孔，放入PCR仪中，设置升温程序为：25℃恒温5min，以0.5℃/min的速度升温至99℃。程序结束后在“Melt Curve”图中读取曲线的最低点的温度值，即为样品的Tm值。具体结果如下表3所示，点突变以及亲和力优化没有降低抗体的热稳定性。
表3.Anti-HER2单克隆抗体的Tm值
实施例6：Anti-HER2抗体的亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于高通量法的抗体-抗原亲和力和表位结合的测定.MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的人或食蟹猴的HER2抗原(购自AcroBiosystems)中作用5min，之后将传感器转移至分析缓冲液中解离5min，最后使用1:1结合模型进行动力学的分析。
实验结果如图2A和图2B所示，本发明中优化的Anti-HER2抗体的亲和力与优化前的Trastuzumab相当。Trastuzumab PTM removal单臂抗体的亲和力明显弱于Trastuzumab单臂抗体，而PTM改造并进行亲和力优化后的本发明的Anti-HER2单臂抗体与人HER2抗原结合的亲和力不亚于Trastuzumab单臂抗体。
实施例7：单克隆抗体与人HER2结合
本实验将扩大培养的N87细胞(自身表达HER2)细胞用0.25％EDTA  trypsin消化，用培养基清洗一次后调整细胞密度至2×106细胞/ml,100μl/孔加入96孔流式板，离心备用。将梯度稀释后的抗体按100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育60min。PBS清洗两次后，100μl/孔加入用2％BSA溶液稀释1000倍的Goat anti-human IgG-Fc(PE)(Abcam,ab98596)，4℃孵育60min。PBS清洗两次，最后按100μl/孔加入PBS重悬细胞，在CytoFlex(Beckman)流式细胞仪上进行检测并计算对应的平均荧光强度(MFI)。
实验结果如图3所示，本发明中优化后的Anti-HER2抗体与人胃癌细胞N87上表达的HER2的结合活性与Trastuzumab相当。Trastuzumab PTM removal单臂抗体的细胞结合活性明显弱于Trastuzumab单臂抗体，而亲和力优化后的Anti-HER2单臂抗体与N87上的HER2抗原结合的亲和力不亚于Trastuzumab单臂抗体，且与单抗相当。
实施例8.Anti-HER2抗体阻断HER2信号依赖的细胞增殖
本实验将扩大培养的N87(自身表达HER2)细胞用0.25％EDTA trypsin消化，用培养基清洗一次后调整细胞密度至5×104细胞/ml，80μl/孔加入96孔板中，备用。将梯度稀释后的抗体按80μl/孔加入上述带有细胞的96孔板中，置于细胞培养箱中孵育3-5天。最后用Luminescent Cell Viability Assay(Promega,G7572)试剂盒显色后用酶标仪收集化学发光信号。
结果如图4所示，本发明中优化后的Anti-HER2单克隆抗体均能够明显抑制N87细胞的增殖，且细胞增殖抑制作用与Trastuzumab单克隆抗体相当。
实施例9.Anti-HER2抗体诱导ADCC效应(报告基因)
本实验将扩大培养的N87(自身表达HER2)细胞细胞按照3×104个/孔与1.2×105个/孔的NFAT Luciferase/Jurkat CD16a(过表达CD16a和NFAT-Luc)效应细胞混合接种至96孔细胞培养白底板中，随后将梯度稀释后HER2单克隆抗体加入96孔板中并混匀，置于细胞培养箱中孵育6小时。 使用Bio-Glo luciferase assay system(Promega,G7940)试剂盒显色后用酶标仪收集化学发光信号。
实验结果如图5所示，本发明中优化后的Anti-HER2单克隆抗体都能通过N87细胞上表达的HER2介导ADCC作用，从而激活Jurkat细胞上的CD16a-NFAT信号通路。此外，Anti-HER2(NO3-46)抗体的ADCC效应与Trastuzumab抗体相当。
实施例10.Anti-HER2单克隆抗体的稳定性研究
10.1.Anti-HER2单克隆抗体的稳定性研究的样品处理
纯化后蛋白样品用稀释液稀释到1mg/ml，分装至西林瓶中后分别置于4℃冰箱和40℃培养箱中孵育2周和4周。样品高温处理2周和4周后进行抗体表征分析与活性验证，如电荷异质性分析，PTM分析和活性验证等。
10.2 Anti-HER2抗体高温处理后的电荷异质性分析
采用阳离子交换色谱法(CEX-HPLC)测定Anti-HER2抗体高温处理后的样品的电荷异质性。CEX-HPLC方法如下：流动相A：20mM MES/MES-Na，pH6.7；流动相B：20mM MES/MES-Na+200mM NaCl，pH6.7；色谱条件：检测波长：280nm；柱温：40℃；流速：1ml/min；检测时间：20min；梯度：3-35min，0％B-100％B；色谱柱ProPac WCX-10(4×250mm,10μm)。
实验结果如图6所示，Trastuzumab在高温加速处理后，CEX-HPLC观察到碱性电荷变体和酸性电荷变体显著增加。相较于Trastuzumab，PTM改造并进行亲和力优化后的本发明的Anti-HER2抗体的碱性电荷变体和酸性电荷变体无明显变化，稳定性显著更优。
10.3 Anti-HER2抗体高温处理后的PTM分析
分别取Trastuzumab及改造分子的高温加速样品200μg于1.5ml离心管中，加入100μl蛋白变性液(8M盐酸胍，pH6.0)，加入2μl 1M DTT，37℃水浴30min进行蛋白变性还原。水浴结束后瞬离30s，加入4.4μL 1mol/L的IAM溶液，涡旋30s混匀，瞬离30s后避光放置30min。吸取避光孵育后样品至10kDa超滤管中，添加300μl 20mmol/L His-HCl pH 6.0 的酶切缓冲液，13000rpm/min离心15min，脱盐后3000g反离3min，回收样品并测定蛋白浓度。取40μg脱盐后的蛋白，加入2μl 0.5mg/ml Trypsin/Lys-C mix酶溶液(酶：蛋白比例为1:40)，补充酶切缓冲液至40μl，轻轻涡旋混匀，瞬离数秒，应避免产生气泡，37℃水浴4小时。酶切结束后加入2μl 20％甲酸水溶液终止反应，混匀后13000rpm/min离心5min，取上清，进行RP-UHPLC-MS分析。
实验结果如表4所示，Trastuzumab在LC-Asn-30/HC-Asn-55/HC-Asp-102位点存在脱酰胺和异构化作用，且样品在高温加速处理后脱酰胺和异构化作用呈现一定程度的提高。RP-UHPLC-MS分析结果显示Trastuzumab在LC-Asn-30位点的脱酰胺程度明显升高，HC-Asn-55/HC-Asp-102位点的脱酰胺或异构化作用的变化较小。而相较于Trastuzumab，PTM改造并进行亲和力优化后的本发明的Anti-HER2抗体在这几个位点检测不到脱酰胺或异构化作用，稳定性显著更优。
表4.Anti-HER2抗体高温处理后的PTM分析结果
N/A表示未检测到
10.4 Anti-HER2抗体高温处理后的活性验证
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于高通量法的抗体-抗原亲和力和表位结合的测定.MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的人HER2抗原(购自AcroBiosystems)中作用5min，之后将传感器转移至分析缓冲液中解离5min，最后使用1:1结合模型进行动力学的分析。
实验结果如图7所示，Trastuzumab在高温处理2周和4周后，其与人HER2抗原结合的亲和力呈现明显的下降，且高温处理时间越长其结合活性越弱。相较于Trastuzumab，PTM改造并进行亲和力优化后的本发明的Anti-HER2抗体在高温处理前后其与HER2抗原的结合活性无明显变化，稳定性显著更优。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (22)

  1. 能够特异性结合HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (1)下述3个重链可变区(VH)互补决定区(CDR)：
    (a)VH CDR1，其具有如GFNIKDTY(SEQ ID NO:10)所示的结构；
    (b)VH CDR2，其具有如IYPTQGYT(SEQ ID NO:11)所示的结构；
    (c)VH CDR3，其具有如SRWGGEGFYAMDY(SEQ ID NO:12)所示的结构；
    和/或，
    (2)下述3个轻链可变区(VL)互补决定区(CDR)：
    (d)VL CDR1，其具有如X1X2VQX3A(SEQ ID NO:33)所示的结构；
    (e)VL CDR2，其具有如SAS(SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27)所示的结构；
    (f)VL CDR3，其具有如QQHX4X5TPPT(SEQ ID NO:34)所示的结构；
    其中：
    X1选自氨基酸残基Q、N；
    X2选自氨基酸残基N、Y、S；
    X3选自氨基酸残基G、T；
    X4选自氨基酸残基Y、F、S；
    X5选自氨基酸残基S、M、T。
  2. 权利要求1所述的抗体或其抗原结合片段，其包含：
    如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，
    如SEQ ID NOs:18、22或26任一项所示的VL CDR1，如SEQ ID NOs:19、23或27任一项所示的VL CDR2，以及，如SEQ ID NOs:20、24或28所示的VL CDR3。
  3. 权利要求1-2任一项所述的抗体或其抗原结合片段，其包含：
    (1)如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:18所示的VL CDR1，如SEQ ID NO:19所示的VL CDR2，以及，如SEQ ID NO:20所示的VL CDR3；或者，
    (2)如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:22所示的VL CDR1，如SEQ ID NO:23所示的VL CDR2，以及，如SEQ ID NO:24所示的VL CDR3；或者，
    (3)如SEQ ID NO:10所示的VH CDR1，如SEQ ID NO:11所示的VH CDR2，以及，如SEQ ID NO:12所示的VH CDR3；和，如SEQ ID NO:26所示的VL CDR1，如SEQ ID NO:27所示的VL CDR2，以及，如SEQ ID NO:28所示的VL CDR3。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其包含：
    如SEQ ID NO:9所示的序列或其变体的VH，和，如SEQ ID NOs:17、21或25任一项所示的序列或其变体的VL；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；优选地，所述的置换是保守置换。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其包含：
    (1)如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:17所示的序列的VL；或者，
    (1)如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:21所示的序列的VL；或者，
    (1)如SEQ ID NO:9所示的序列的VH，和，如SEQ ID NO:25所示 的序列的VL。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其进一步包含来源于人免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区；
    优选地，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其进一步包含如SEQ ID NO:29所示的重链恒定区和/或如SEQ ID NO:31所示的轻链恒定区。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
  9. 单臂抗体，其包含：
    (1)第一肽链，所述第一肽链包含3个重链可变区(VH)互补决定区(CDR)，所述3个重链可变区(VH)互补决定区(CDR)如权利要求1-3任一项所限定，
    (2)第二肽链，所述第二肽链包含3个轻链可变区(VL)互补决定区(CDR)，所述3个轻链可变区(VL)互补决定区(CDR)如权利要求1-3任一项所限定，和，
    (3)第三肽链，所述第三肽链能够和所述第一肽链形成二聚体；
    优选地，所述第一肽链包含重链可变区(VH)，所述重链可变区(VH)如权利要求4-5任一项所限定；
    优选地，所述第二肽链包含轻链可变区(VL)，所述轻链可变区(VL)如权利要求4-5任一项所限定。
  10. 权利要求9所述的单臂抗体，其中，所述第二肽链进一步包含来源于人免疫球蛋白的恒定区；
    优选地，所述第二肽链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，所述第二肽链包含如SEQ ID NO:31所示的轻链恒定区。
  11. 权利要求9-10任一项所述的单臂抗体，其中，所述单臂抗体进一步具有选自以下(1)-(2)中至少一项的技术特征：
    (1)所述第一肽链进一步包含来源于人免疫球蛋白的恒定区；优选地，所述来源于人免疫球蛋白的恒定区为来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区；优选地，所述重链恒定区具有第一修饰，以促进所述第一肽链和所述第三肽链的二聚化；
    (2)所述第三肽链包含Fc结构域单体；优选地，所述Fc结构域单体是IgG的Fc结构域单体，例如IgG1、IgG2、IgG3或IgG4的Fc结构域单体；优选地，所述Fc结构域单体具有第二修饰，以促进所述第三肽链和所述第一肽链的二聚化；
    优选地，所述第一修饰和所述第二修饰中，任意一个为“节”修饰，另一个为“穴”修饰，以形成节-入-穴(knob-into-hole)”修饰，促进所述第一肽链和所述第三肽链的二聚化；
    优选地，所述第一修饰为“节”修饰，所述第二修饰为“穴”修饰，以形成节-入-穴(knob-into-hole)”修饰，促进所述第一肽链和所述第三肽链的二聚化；
    优选地，所述重链恒定区包含如SEQ ID NO:30所示的氨基酸序列，所述Fc结构域单体包含如SEQ ID NO:32所示的氨基酸序列。
  12. 分离的核酸分子，其编码权利要求1-8任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；或者，其编码权利要求9-11任一项所述的单臂抗体，或其重链可变区和/或轻链可变区。
  13. 载体，其包含权利要求12所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  14. 宿主细胞，其包含权利要求12所述的核酸分子或权利要求13所述的载体。
  15. 制备权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体的方法，其包括，在允许所述抗体或其抗原结合片段或者所述单臂抗体表达的条件下，培养权利要求14所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段或者所述单臂抗体。
  16. 药物组合物，其包含权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体，以及任选的药学上可接受的载体和/或赋形剂。
  17. 权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9-11任一项所述的单臂抗体、权利要求12所述的分离的核酸分子、权利要求13所述的载体或权利要求14所述的宿主细胞用于制备药物的用途，所述药物用于在受试者中激活HER2，提高免疫细胞活性，增强免疫应答，和/或预防和/或治疗肿瘤或者感染；
    优选地，所述免疫细胞是T细胞，B细胞，DC细胞，巨噬细胞，和/或，NK细胞；
    优选地，所述免疫应答是HER2介导的免疫应答；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述抗体或其抗原结合片段或者所述单臂抗体单独使用，或与另外的药学活性剂联合使用。
  18. 一种用于在受试者中增强免疫应答，和/或，预防和/或治疗肿瘤或 感染的方法，所述方法包括：给有此需要的受试者施用有效量的权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体或者权利要求16所述的药物组合物；
    优选地，所述免疫应答是HER2介导的免疫应答；
    优选地，所述受试者为哺乳动物，例如人。
  19. 缀合物，其包含权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体，以及任选的与所述抗体或其抗原结合片段或者所述单臂抗体连接的可检测的标记；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  20. 试剂盒，其包含权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体或者权利要求19所述的缀合物；
    优选地，所述试剂盒包含权利要求19所述的缀合物；
    优选地，所述试剂盒包含权利要求1-8任一项所述的抗体或其抗原结合片段，以及特异性识别所述抗体或其抗原结合片段的第二抗体；任选地，所述第二抗体还包含可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素；
    优选地，所述试剂盒包含权利要求9-11任一项所述的单臂抗体，以及特异性识别所述单臂抗体的第二抗体；任选地，所述第二抗体还包含可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  21. 用于检测HER2在样品中的存在或其水平的方法，其包括使用权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述 的单臂抗体或者权利要求19所述的缀合物；
    优选地，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述方法包括使用权利要求19所述的缀合物；
    优选地，所述方法包括使用权利要求1-8任一项所述的抗体或其抗原结合片段，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述抗体或其抗原结合片段；
    优选地，所述方法包括使用权利要求9-11任一项所述的单臂抗体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述单臂抗体。
  22. 权利要求1-8任一项所述的抗体或其抗原结合片段或者权利要求9-11任一项所述的单臂抗体或者权利要求19所述的缀合物在制备检测试剂中的用途，所述检测试剂用于检测HER2在样品中的存在或其水平；
    优选地，所述检测试剂通过权利要求21所述的方法来检测HER2在样品中的存在或其水平；
    优选地，所述样品为来自受试者(例如哺乳动物，优选人或食蟹猴)的细胞样品(例如，免疫细胞)。
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