WO2022121969A1 - Gpc3抗体及其应用 - Google Patents

Gpc3抗体及其应用 Download PDF

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WO2022121969A1
WO2022121969A1 PCT/CN2021/136637 CN2021136637W WO2022121969A1 WO 2022121969 A1 WO2022121969 A1 WO 2022121969A1 CN 2021136637 W CN2021136637 W CN 2021136637W WO 2022121969 A1 WO2022121969 A1 WO 2022121969A1
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antibody
antigen
cancer
gpc3
cells
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PCT/CN2021/136637
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English (en)
French (fr)
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游术梅
杨翠青
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to CN202180082576.5A priority Critical patent/CN116670287A/zh
Priority to US18/266,393 priority patent/US20240052055A1/en
Publication of WO2022121969A1 publication Critical patent/WO2022121969A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to the field of antibodies, in particular, to GPC3 antibodies and applications thereof.
  • Glypican-3 is a heparan sulfate (HS) glycoprotein, a member of the heparan sulfate proteoglycan family, which is anchored to the cell membrane by Glypican-3 (GPI) surface.
  • the GPC3 core protein includes 580 amino acids and is about 70KD in size. After it is cleaved by Furin, a 40kD amino (N) terminal subunit and a 30kD carboxyl (C) terminal subunit are generated. Disulfide linkages.
  • the two HS side chains of GPC3 bind close to the C-terminus (Takahiro Nishida, Hiroaki Kataoka. Glypican 3-Targeted Therapy in Hepatocellular Carcinoma, Cancers 2019; 11(9):1339).
  • GPC3 plays an important regulatory role in cell proliferation in embryonic mesoderm tissue, and deletion of the GPC3 gene results in an overgrowth syndrome, Simpson-Golabi-Behmel syndrome (SGBS).
  • GPC3 was significantly expressed throughout the fetal period, but was not significantly expressed in other normal tissues except for the weak expression in placenta, mammary gland, mesothelial, ovary, lung and kidney tissues after birth to adulthood.
  • GPC3 is abnormally expressed in various adult tumor tissues, such as hepatocellular carcinoma (HCC), lung squamous cell carcinoma, gastric cancer, ovarian cancer, etc.
  • HCC hepatocellular carcinoma
  • lung squamous cell carcinoma gastric cancer
  • ovarian cancer etc.
  • the obtained single domain antibody (single domain antibody, sdAb) composed of only one heavy chain variable region is called nanobody or VHH domain (variable heavy chain domain of a) heavy chain antibody).
  • nanobodies Compared with ordinary antibodies, nanobodies have many advantages such as small molecular weight, good stability and high solubility. Therefore, the application of nanobody technology to develop GPC3 antibodies has broad prospects, and obtaining GPC3-specific nanobodies is of great significance for the development of related therapeutic drugs or detection reagents.
  • the present disclosure discloses antibodies or antigen-binding fragments, polypeptides, chimeric antigen receptors, immune effector cells, isolated nucleic acid fragments, vectors, host cells, corresponding preparation methods, pharmaceutical compositions, treatment methods, pharmaceutical uses, which specifically bind to GPC3, GPC3 detection method and detection kit.
  • the present disclosure relates to an antibody or antigen-binding fragment that specifically binds GPC3, the antibody or antigen-binding fragment comprising CDRl, CDR2, and CDR3.
  • the CDR1, CDR2 and CDR3 are HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system, for example, selected from Table 1; for example, the HCDR1 is selected from SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 88, 91 or 94, the HCDR2 is selected from the group consisting of SEQ ID: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 89, 92 or 95, and said HCDR3 is selected from SEQ ID: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 24-26, 27-29 or 30-32;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 33-35, 36-38 or 39-41;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 42-44, 45-47 or 48-50;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 51-53, 54-56 or 57-59;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 60-62, 63-65 or 66-68;
  • the HCDR1, HCDR2 and HCDR3 are selected from the sequences of SEQ ID NOs: 69-71, 72-74 or 75-77 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 78-80, 81-83 or 84-86;
  • the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NOs: 88-90, 91-93 or 94-96 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.
  • said CDR1, CDR2 and/or CDR3 comprises at most 10, 9, 8, 7, 6, 5, 4 occurrences on said HCDR1, HCDR2 and/or HCDR3 1, 3, 2 or 1 mutated amino acid sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably the substitutions are conservative amino acid substitutions.
  • said CDR1, CDR2 and/or CDR3 comprise at least 80%, 85%, 90%, 91%, 92%, 93%, Sequences of 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment comprises a single domain antibody comprising the CDRl, CDR2 and CDR3.
  • the single-domain antibody comprises the sequence shown in any one of SEQ ID NOs: 17-23 or 87; alternatively, the single-domain antibody comprises the same sequence as SEQ ID NO: 17-23 or 87 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity compared to any one of the indicated sequences or the single-domain antibody comprises at most 20, 19, 18, 17, 16, 15, 14 compared with the sequence shown in any one of SEQ ID NOs: 17-23 or 87 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutated sequence, which may be selected from insertion , deletions and/or substitutions, preferably conservative amino acid substitutions.
  • the single-domain antibody comprises a FR region in the VHH domain shown in any one of SEQ ID NOs: 17-23 or 87; alternatively, the single-domain antibody comprises a FR region with SEQ ID NO : at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% compared to the FR regions in the VHH domains shown in any one of 17 to 23 or 87 %, 98%, 99% or 100% identical sequences, or the single domain antibody comprises at most 15 FR regions compared to the FR regions in the VHH domain set forth in any one of SEQ ID NOs: 17-23 or 87 , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutated sequences that may is selected from insertions, deletions and/or substitutions, preferably conservative amino acid substitutions.
  • the antibody or antigen-binding fragment is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.
  • the antibody or antigen-binding fragment includes or does not include an antibody heavy chain constant region; alternatively, the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat, Rabbit or sheep; alternatively, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain The constant region can be selected from Fc region, CH3 region or complete heavy chain constant region, preferably, the heavy chain constant region is a human Fc region, more preferably has the amino acid sequence shown in SEQ ID NO: 1; preferably, the antibody Or the antigen-binding fragment is a heavy chain antibody.
  • the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat, Rabbit or sheep; alternatively, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE or IgD, and the I
  • the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents
  • the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the VHH domain specifically binds to human, monkey and/or murine GPC3; preferably, its KD for binding to human and/or monkey GPC3 is better than 1.00E-7M, 1.00E-8M , 2.00E-8M, 3.00E-8M, 4.00E-8M, 5.00E-8M, 6.00E-8M, 7.00E-8M, 8.00E-8M, 9.00E-8M, 1.00E-9M, 2.00E-9M , 3.00E-9M, 4.00E-9M, 5.00E-9M, 6.00E-9M, 7.00E-9M, 8.00E-9M, 9.00E-9M or 1.00E-10M.
  • the present disclosure discloses an antibody or antigen-binding fragment that specifically binds GPC3, the antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3; the CDR1, CDR2 and CDR3 comprising a group selected from SEQ ID NO: 17 HCDR1 , HCDR2 and HCDR3 in the VHH domains set forth in any of ⁇ 23 or 87.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system, for example, selected from Table 1; for example, the HCDR1 is selected from SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 88, 91 or 94, the HCDR2 is selected from the group consisting of SEQ ID: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 89, 92 or 95, and said HCDR3 is selected from SEQ ID: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 24-26, 27-29 or 30-32;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 33-35, 36-38 or 39-41;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 42-44, 45-47 or 48-50;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 51-53, 54-56 or 57-59;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 60-62, 63-65 or 66-68;
  • the HCDR1, HCDR2 and HCDR3 are selected from the sequences of SEQ ID NOs: 69-71, 72-74 or 75-77 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system;
  • the Kabat numbering system or the Chothia numbering system are selected from the sequences of SEQ ID NOs: 78-80, 81-83 or 84-86;
  • the HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NOs: 88-90, 91-93 or 94-96 according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system. .
  • said CDR1, CDR2 and/or CDR3 comprises at most 10, 9, 8, 7, 6, 5, 4 occurrences on said HCDR1, HCDR2 and/or HCDR3 1, 3, 2 or 1 mutated amino acid sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably the substitutions are conservative amino acid substitutions.
  • said CDR1, CDR2 and/or CDR3 comprise at least 80%, 85%, 90%, 91%, 92%, 93%, Sequences of 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the present disclosure also discloses a polypeptide, the polypeptide comprises the aforementioned antibody or antigen-binding fragment, preferably, the polypeptide is further linked with other functional molecules, and the other functional molecules can be selected from one of the following or more: signal peptides, protein tags, other antigen binding molecules or cytokines.
  • the other antigen-binding molecule specifically binds an antigen other than GPC3 or binds a different GPC3 epitope than the aforementioned antibody or antigen-binding fragment;
  • the antigens other than GPC3 can be selected from: CD3, preferably CD3 ⁇ ; CD16, preferably CD16A; NKG2D; CD40; 4-1BB; CD137 or CD19; EGFR; EGFRvIII; mesothelin; HER2; EphA2; Her3; EpCAM; MUC1 ; MUC16; CEA; Claudin18.2; folate receptor; Claudin6; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16;
  • the other antigen-binding molecules are antibodies or antigen-binding fragments
  • the polypeptide is a multispecific antigen binding molecule
  • the multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific, more preferably, the multispecific antigen binding molecule may be Two, four or six.
  • the cytokine may be selected from IL2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha.
  • the present disclosure also discloses a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the cell
  • the outer antigen-binding domain comprises the aforementioned antibodies or antigen-binding fragments.
  • the present disclosure also discloses an immune effector cell, the immune effector cell expressing the aforementioned chimeric antigen receptor, or comprising a nucleic acid fragment encoding the aforementioned chimeric antigen receptor; preferably, the immune effector cell selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, the T cells
  • the cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present disclosure also discloses an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment, polypeptide, or chimeric antigen receptor.
  • the present disclosure also discloses a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also discloses a host cell, the host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insects cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insects cells or mammalian cells (CHO cell line or 293T cell line).
  • the present disclosure also discloses a method for preparing the aforementioned antibody or antigen-binding fragment or polypeptide, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell, or isolating the cell-expressed antibody or antigen-binding fragment of polypeptides.
  • the present disclosure further discloses a method for preparing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, optionally, the method further comprising activating the immune effector cells
  • the effector cells express the aforementioned CAR.
  • the present disclosure also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment, polypeptide, immune effector cell, nucleic acid fragment, carrier or product prepared according to the aforementioned method;
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant;
  • the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present disclosure also discloses a method of treating a GPC3-positive tumor or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibodies or antigen-binding fragments, polypeptides, immune effector cells, nucleic acid fragments, A carrier, a product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition;
  • the GPC3-positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, and renal cancer , pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, schwannoma, malignant fibrous histiocytoma or esophageal cancer; more preferably, the GPC3-positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer or breast cancer .
  • the present disclosure further discloses that the aforementioned antibodies or antigen-binding fragments, polypeptides, immune effector cells, nucleic acid fragments, vectors, products prepared according to the aforementioned methods, or the aforementioned pharmaceutical compositions are used in the preparation of drugs for the treatment of GPC3-positive tumors or cancers
  • the GPC3-positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma tumor, adrenal carcinoma, schwannoma, malignant fibrous histiocytoma or esophageal carcinoma; more preferably, the GPC3 positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer or breast cancer.
  • the present disclosure also discloses the aforementioned antibodies or antigen-binding fragments, polypeptides, immune effector cells, nucleic acid fragments, vectors, products prepared according to the aforementioned methods, or the aforementioned pharmaceutical compositions for the treatment of GPC3-positive tumors or Cancer; preferably, the GPC3-positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal cancer, schwannoma, malignant fibrous histiocytoma or esophageal cancer; more preferably, the GPC3 positive tumor or cancer is selected from liver cancer, gastric cancer, lung cancer or breast cancer.
  • the present disclosure further discloses a kit comprising the aforementioned antibody or antigen-binding fragment, polypeptide, immune effector cell, nucleic acid fragment, vector, host cell, product prepared according to the aforementioned method, or The aforementioned pharmaceutical composition.
  • the present disclosure also discloses a method for detecting the expression of GPC3 in a biological sample, the method comprising causing the biological sample to form a complex between the aforementioned antibody or antigen-binding fragment and GPC3 under conditions that allow the biological sample to form a complex.
  • the chemical sample is contacted with the aforementioned antibody or antigen-binding fragment; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of GPC3 in the sample.
  • the present disclosure also discloses the use of the aforementioned antibody or antigen-binding fragment in the preparation of a GPC3 detection reagent.
  • the present disclosure further discloses a kit for detecting GPC3, the kit comprising at least the aforementioned antibody or antigen-binding fragment.
  • Glypican-3 herein is a heparan sulfate (HS) glycoprotein, a member of the heparan sulfate proteoglycan family, which is mediated by Glypican-3 (GPI) anchored to the cell membrane surface.
  • GPC3 herein includes mature or immature full-length wild-type GPC3 protein or mutants thereof (eg, point mutations, insertion mutations or deletion mutations), splice variants, orthologs (Orthologs) and the foregoing Fragment of GPC3.
  • GPC3 can be derived from mammals, eg, humans, primates, such as monkeys (eg, rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats.
  • primates such as monkeys (eg, rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats.
  • amino acid sequence of human GPC3 can be found in NCBI: NM_004484.3
  • the amino acid sequence of monkey GPC3 can be found in NCBI: XP_011739317.1.
  • the term "specifically binds" herein refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • high affinity generally refers to having about 10-7 M or less, about 10-8 M or less, about 1 ⁇ 10-9 M or less, about 1 ⁇ 10-10 M or less, KD of 1 ⁇ 10-11 M or lower or 1 ⁇ 10-12 M or lower.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
  • antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is The N-terminal to C-terminal direction consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, the hinge region (HR), the heavy chain constant region CH2 domain, the heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction
  • the polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure.
  • immunoglobulins Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ and ⁇ chains, respectively. , ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other camelids produced heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin receptor (Ig new antigen receptor, IgNAR).
  • HCAbs heavy-chain antibodies
  • the term “heavy chain antibody” refers to an antibody that lacks the light chain of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising the VH antigen binding domain and the CH2 and CH3 constant domains in the absence of the CH1 domain.
  • VHH domain and “nanobody” and “single domain antibody” (sdAb) herein have the same meaning and can be used interchangeably, and refer to the variable region of cloning heavy chain antibodies, constructing A single-domain antibody consisting of only one heavy chain variable region, it is the smallest fully functional antigen-binding fragment.
  • a heavy chain antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • an “antibody” herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • multispecific herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
  • the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
  • full-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean having a structure that is substantially similar to that of a native antibody.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein and do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody that have the ability to bind antigenic capacity.
  • antigen-binding fragments or “antibody fragments” herein include, but are not limited to, Fab, F(ab') 2 , Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domains Antibody.
  • chimeric antibody herein refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat, mouse, rabbit, or alpaca) and derived from a different organism (eg, human ) of the constant regions of immunoglobulins.
  • Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, Bio Techniques 4: 214-221; Gillies et al, 1985 J Immunol Methods 125: 191-202; incorporated by reference above This article.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
  • Fully human antibody refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
  • "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”.
  • VL is used interchangeably with "VL”, “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • the light chain variable region CDR can be abbreviated as LCDR.
  • framework region or "FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody.
  • CDRs may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues differently defined.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • IMGT International ImMunoGeneTics information system
  • S001-NB150-20 (cyno+), S001-NB150-28 (cyno+), S001-NB150-56, S001-NB152-1, S001-NB152-101, S001-NB152 were determined using Kabat, Chothia and IMGT CDRs of -72-2, VH001 (SEQ ID NO: 17 ⁇ 23), VHH-12 (SEQ ID NO: 87), the specific results are shown in Table 1:
  • heavy chain constant region herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
  • the variable domains have more conserved amino acid sequences.
  • the “heavy chain constant region” may be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage, cleavage of one or more, particularly one or two amino acids, from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region comprises the IgG CH2 and IgG CH3 domains, optionally, the entire or partial hinge region, but not the CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • a CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain.
  • the "CH3 domain" comprises that stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • a CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole", hole) in the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • a variant CH3 domain eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole”, hole) in the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National As described in Institutes of Health, Bethesda, MD, 1991.
  • amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • identity herein can be calculated by aligning the sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (eg, it may be optimal). The alignment may introduce gaps in either or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences varies with the identical positions shared by the sequences.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com) using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences.
  • a particularly preferred set of parameters is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described in this disclosure can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • Such searches can be performed, for example, using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is generally represented as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg, cDNA
  • RNA eg, mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823B1).
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • pharmaceutical composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.
  • treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) undesired physiological changes or pathologies, such as cancers and tumors, in a subject being treated.
  • Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • subject herein refers to an organism receiving treatment for a particular disease or disorder as described in the present disclosure.
  • a “subject” includes a mammal, such as a human, primate (eg, monkey) or non-primate mammal, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
  • Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • GPC3-positive refers to a cell or tissue whose cellular expression of GPC3 is higher than the normal expression level of the corresponding cell or tissue, and “GPC3-positive” can be detected by methods known in the art.
  • Fig. 1A shows the binding reaction of ELISA detection control antibody and human GPC3-His protein
  • Fig. 1B shows the binding reaction of ELISA detection control antibody and monkey GPC3-His protein
  • Figure 1C shows the binding reaction of ELISA detection control antibody and mouse GPC3-His protein
  • Fig. 2A is the binding reaction of ELISA detection polypeptide GC3pep protein and control antibody
  • Fig. 2B is the binding reaction of ELISA detection polypeptide YP7pep protein and control antibody;
  • Figure 3A is the FACS result of the Tab003 antibody detecting the expression of GPC3 in HepG2 cells
  • Figure 3B is the FACS result of the Tab005 antibody detecting the expression of GPC3 in HepG2 cells
  • Figure 4A is the FACS result of the Tab003 antibody detecting the expression of GPC3 in CHOK1-hGPC3.1C3 cells;
  • Figure 4B is the FACS result of the Tab003 antibody detecting GPC3 expression in CHOK1-hGPC3.2B5 cells;
  • Figure 4C is the FACS result of the Tab003 antibody detecting GPC3 expression in CHOK1-hGPC3.3E9 cells;
  • Figure 5 is the FACS results of the Tab003 antibody detecting the expression of GPC3 in HEK293T-monkey GPC3 cells
  • Figure 6A shows the situation of the serum antibody titer of alpaca after human GPC3 protein immunization detected by ELISA
  • Figure 6B shows the serum antibody titer of alpaca after human GPC3 protein immunization detected by FACS
  • Figures 7A-7B show the binding reaction of VHH-hFc and human GPC3-his protein detected by ELISA
  • Fig. 8A is FACS detection of the binding reaction of VHH-hFc and CHO-K1-human GPC3 cells;
  • Figure 8B shows the binding reaction of VHH-hFc to CHO-K1 cells detected by FACS
  • Figure 9A and Figure 9C are FACS detection of the binding reaction of VHH-hFc and HepG2 tumor cells
  • Figure 9B and Figure 9D are FACS detection of the binding reaction of VHH-hFc to A431 tumor cells
  • Figure 10 shows the binding reaction of VHH-hFc and monkey GPC3-His protein detected by ELISA
  • Figure 11 shows the binding reaction of VHH-hFc and murine GPC3-his protein detected by ELISA
  • Figure 12A shows the binding reaction of VHH-hFc and HEK293T-monkey GPC3 cells detected by ELISA
  • Figure 12B shows the binding reaction of VHH-hFc and HEK293T cells detected by ELISA
  • Figure 13A shows the binding reaction of VHH-hFc and GC3pep polypeptide protein detected by ELISA
  • Figure 13B shows the binding reaction of VHH-hFc and YP7pep polypeptide protein detected by ELISA.
  • Example 1 Preparation of control antibodies, preparation of human polypeptides, identification of endogenous cells and preparation of overexpressing cell lines
  • VL and VH of the monoclonal antibodies Y035 and T2-23 that recognize human GPC3 and the human IgG1Fc are connected in the sequence from the N-terminus to the C-terminus, wherein the VH and VL are connected by 3 GGGGS linkers to form scFv-human IgG1Fc (scFv-hFc), the corresponding nucleotide sequences were cloned into pTT5 vector (purchased from Youbao Bio), and plasmids were prepared according to established standard molecular biology methods. For specific methods, see Sambrook, J. , Fritsch, E.F., and Maniatis, T. (1989).
  • the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borglon), washed with PBS phosphate buffer (pH 7.4) Washed with 20mM PB, 1M NaCl, pH 7.2, and finally eluted with pH 3.4 citrate buffer to collect the Fc-tagged antibody eluted from the Protein A column, with 1/10 volume of Neutralized with 1M Tris at pH 8.0, dialyzed with PBS overnight at 4°C, the concentration of the dialyzed antibody was determined by Nanodrop, the purity of the antibody was determined by HPLC-SEC, and the antibody was detected by endotoxin detection kit (purchased from Andus).
  • GC3pep AELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO:8); YP7pep: DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS (SEQ ID NO:9).
  • the prepared polypeptides were tested by ELISA with positive control antibodies that recognize different epitopes.
  • the test results are shown in Tables 6-7 and Figures 2A and 2B.
  • Tab005 could not bind to the polypeptides GC3pep and YP7pep
  • Tab003 could bind to the polypeptides GC3pep and YP7pep.
  • the detection results are consistent with those reported in the literature, indicating that the above-mentioned polypeptides with binding activity have been prepared and obtained.
  • Table 7 ELISA detects the binding reaction of control antibody and polypeptide YP7pep protein
  • HepG2 cells were expanded and cultured in a T-75 cell culture flask to the logarithmic growth phase, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • Tab003 and Tab005 antibodies were used as primary antibodies, and FITC-labeled secondary antibodies (purchased from Invitrogen, product number: A11013) were detected and analyzed by FACS (FACS CantoTM, purchased from BD Company). The results are shown in Table 8 and Figures 3A-3B, indicating that HepG2 cells can bind to both Tab003 and Tab005.
  • the nucleotide sequence encoding the full-length amino acid sequence of human GPC3 was cloned into pcDNA3.1 vector (purchased from Clontech) and a plasmid was prepared.
  • the CHO-K1 cell line purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences
  • plasmids 3000 Transfection Kit, purchased from Invitrogen, Cat. No. L3000-015
  • DMEM/F12 medium containing 10% (w/w) fetal bovine serum containing 10 ⁇ g/mL puromycin, and treated with Tab003 antibody.
  • Table 9 illustrates that a series of human GPC3-positive CHO-K1 monoclonal cell lines have been generated.
  • the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
  • the nucleotide sequence encoding the monkey GPC3 full-length amino acid sequence (NCBI: XP_011739317.1, SEQ ID NO: 11) was cloned into pcDNA3.1 vector (purchased from Thermofisher scientific) and a plasmid was prepared.
  • pcDNA3.1 vector purchased from Thermofisher scientific
  • a plasmid was prepared.
  • HEK293T cell line HD Promega, Cat. No.: #E2311
  • was selectively cultured for 2 weeks in DMEM medium containing 10 ⁇ g/mL puromycin and 10% (w/w) fetal bovine serum after plasmid transfection with Tab003 antibody and donkey Anti-human IgG H+L antibody (Jackson, Cat.
  • alpacas Two alpacas (Alpaca, NB150 and NB152) were immunized with human GPC3 (Gln 25-His 559)-His protein (purchased from Acro, catalog number: GP3-H52H4) and polypeptide GC3pep-KLH.
  • human GPC3-His protein was emulsified with Freund's complete adjuvant and then injected subcutaneously at multiple points, namely, 500 ⁇ g of human GPC3-His protein was injected into each alpaca.
  • human GPC3-His protein was emulsified with incomplete Freund's adjuvant and then injected subcutaneously at multiple points, namely, 250 ⁇ g of human GPC3-His protein was injected into each alpaca.
  • the polypeptide GC3pep-KLH was emulsified with incomplete Freund's adjuvant and then injected subcutaneously at multiple points, that is, 250 ⁇ g of the polypeptide GC3pep-KLH was injected into each alpaca.
  • Table 10 ELISA detects the antibody titer of alpaca serum after human GPC3-His protein immunization
  • RNAiso Plus reagent The extracted RNA was reverse transcribed into cDNA using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (purchased from Takara, Cat. No. 6210A). Amplification of variable region nucleic acid fragments encoding heavy chain antibodies by nested PCR:
  • Upstream primer (SEQ ID NO: 12): CTTGGTGGTCCTGGCTGC;
  • Downstream primer (SEQ ID NO: 13): GGTACGTGCTGTTGAACTGTTCC.
  • Upstream primer SEQ ID NO: 14:
  • the target single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pcomb3XSS using the restriction enzyme SfiI.
  • the product was then electro-transformed into E. coli electro-competent cells TG1, and a single-domain antibody phage display library against GPC3 was constructed and assayed.
  • the size of the library volume was calculated to be 3.4 ⁇ 10 9 .
  • 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
  • the human GPC3-Llama-Fc protein (purchased from Acro, product number: GP3-H5257) was diluted with a carbonate buffer with a pH value of 9.6 to a final concentration of 5 ⁇ g/mL, and added to the enzyme-labeled well at 100 ⁇ L/well.
  • Dilute human GPC3 protein and GC3pep polypeptide with carbonate buffer with pH 9.6 to a final concentration of 2 ⁇ g/mL add 100 ⁇ L wells to enzyme-labeled wells, and coat overnight at 4°C; discard the coating solution and wash with PBST 3 times, add 300 ⁇ L 5% skim milk to each well, block at 37°C for 1 hour; wash 3 times with PBST, add 50 ⁇ L phage culture supernatant and 50 ⁇ L 5% skim milk to each well, incubate at 37°C for 1 hour; wash 5 times with PBST, add Horseradish peroxidase-labeled anti-M13 antibody (diluted at 1:10000 in PBS), 100 ⁇ L/well, reacted at 37° C.
  • the sequencing results were analyzed, and the phylogenetic tree was constructed according to the VHH-encoded protein sequence, and the sequences that were closer in the phylogenetic tree were eliminated according to the sequence similarity to identify the production of VHH-hFc.
  • VHH sequences used for production identification are shown below and based on the IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results), the abYsis website (www.abysis.org/abysis/ sequence_input/key_annotation/key_annotation.cgi) and AbRSA website (http://cao.labshare.cn/AbRSA/cdrs.php) to determine the CDR region of the VHH sequence, see Table 1 for details.
  • the target VHH sequence was recombined into the expression vector of human IgG1Fc to obtain a recombinant plasmid.
  • the specific plasmid construction, transfection and purification procedures refer to Example 1(A).
  • the purified VHH-hFc was analyzed for protein concentration, purity, and endotoxin (Lonza kit). The results are shown in Table 11. It was found that the final product of the antibody had a high purity, and the endotoxin concentration was within 1.0 EU/mg.
  • Human GPC3 protein was diluted with PBS to a final concentration of 1 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ l per well. Cover with plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/w) BSA] and block for 2 hours at room temperature. Pour off the blocking solution and add 100 nM serially diluted VHH-hFc, positive control antibody or negative control antibody, 50 ⁇ l per well. After incubation at 37°C for 2 hours, the plate was washed 3 times with PBS.
  • the S001-NB152-1 antibody had weak binding to human GPC3 protein, and the other seven VHH-hFcs were well bound to human GPC3 protein.
  • the IgG control is hIgG1, and the data in the table is the OD450nm value.
  • Table 12-1 ELISA detects the binding reaction of VHH-hFc and human GPC3 protein
  • Table 12-2 ELISA detects the binding reaction of VHH-hFc and human GPC3 protein
  • the desired cells were expanded to logarithmic growth phase in T-75 cell culture flasks, the medium was aspirated, washed twice with PBS buffer, cells were trypsinized, then the digestion was terminated with complete medium, and cells were pipetted to single-cell suspension. After cell counting, centrifuge, resuspend the cell pellet with FACS buffer (PBS+2% fetal bovine serum) to 2 ⁇ 10 6 cells per ml, add 50 ⁇ l per well to a 96-well FACS reaction plate, add VHH-hFc, positive Control antibody or negative control antibody, 50 ⁇ l per well, incubated at 4 degrees for 1 hour.
  • FACS buffer PBS+2% fetal bovine serum
  • the cells were centrifuged and washed three times with PBS buffer, 50 ⁇ l of secondary antibody anti-hlgG(H+L) Alexa 647 (purchased from Jackson, catalog number: 109-605-088) was added to each well, and incubated on ice for 1 hour.
  • the cells were centrifuged and washed 3 times with PBS buffer, and 100 ⁇ l was detected and analyzed by FACS (FACS CantoTM, purchased from BD Company). Data analysis was performed by software (FlowJo) to obtain the mean fluorescence intensity (MFI) of the cells. Then, it was analyzed by software (GraphPad Prism8), data fitting was performed, and EC50 was calculated.
  • Tables 13-14, Figures 8A-8B, and Figures 9A-9D show that both VHH-hFc can bind to CHO-K1-human GPC3 cells, do not bind to CHO-K1 cells;
  • Table 14-1, Table 14-2 and Figure 9A-9D show that VHH-hFc can bind to HepG2 cells, except for the antibody S001-NB150-20 (cyno+), other antibodies are not bound A431 cells.
  • the monkey GPC3-His protein (purchased from Acro, product number: GP3-C5225) was subjected to ELISA detection and data analysis according to the method of Example 4(A).
  • the ELISA results of VHH-hFc and monkey GPC3 protein are shown in Figure 10 and Table 15. The results show that S001-NB150-56 antibody has weak binding to monkey GPC3 protein, S001-NB152-1 antibody does not bind to monkey GPC3 protein, and the rest VHH-hFc binds well to monkey GPC3 protein.
  • the IgG control is hIgG1
  • the data in the table is the OD450nm value.
  • the murine GPC3-his protein (purchased from Sino Biological, product number: 50989-M08B) was subjected to ELISA detection and data analysis according to the method of Example 4(A).
  • the ELISA results of VHH-hFc and mouse GPC3 protein are shown in Figure 11 and Table 16. The results show that S001-NB152-1 antibody has weak binding to mouse GPC3 protein, S001-NB150-20 (cyno+) and S001-NB152-101 It does not bind to mouse GPC3 protein, and the other four VHH-hFc bind well to mouse GPC3 protein.
  • the HEK293T-monkey GPC3 cells were subjected to FACS detection and data analysis according to the method of Example 4(B). The analysis results are shown in Table 17 and Figures 12A and 12B. All VHH-hFc bound to HEK293T-monkey-GPC3 cells, but did not bind to HEK293T cells.
  • Anti-human GPC3 VHH-hFc was captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25 °C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, and then a single concentration of GPC3 antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein.
  • the binding rate (Ka), dissociation rate (Kdis) and binding affinity (KD) of VHH-hFc to human GPC3 protein are shown in the table, in which the antibody Tab003 is used as a control.
  • the affinity of VHH-hFc to human GPC3 protein was better than that of 1E-7M, S001-NB150-28(cyno+), S001-NB152-101 and S001 -The affinity of NB152-72-2 and VHH-12 to human GPC3 protein is close to 1E-8M, the affinity of other VHH-hFc to human GPC3 protein is better than that of 1E-8M, and the affinity of VH001 is close to 1E-9M.
  • VHH-hFc and monkey GPC3-His protein The affinity detection of VHH-hFc and monkey GPC3-His protein was carried out according to the method of Example 6(A), in which the antibody Tab003 was used as a control. As shown in Table 19, the affinity of VHH-hFc to monkey GPC3 protein was better than that of 1E-8M.
  • VHH-hFc and murine GPC3-His protein were carried out according to the method of Example 6(A), in which the antibody Tab005 was used as a control.
  • S001-NB150-20(cyno+) and S001-NB152-101 did not bind to mouse GPC3 protein, and the other five VHH-hFcs had better affinities to mouse GPC3 protein than 1E-7M, among which VH001 and mouse GPC3 protein
  • the binding affinity of GPC3 protein is around 1E-8M, and the affinity of S001-NB150-28(cyno+), S001-NB150-56, S001-NB152-1 and S001-NB152-72-2 is above 1E-8M.
  • the mature GPC3 protein has a soluble amino-terminal (N-terminal) peptide of about 40 kD and a membrane-bound carboxyl-terminal (C-terminal) peptide of about 30 kD that can enter the blood.
  • the Tab003 antibody recognizes the C-terminal region of GPC3 protein close to the cell membrane (proximal end), and the Tab005 antibody recognizes the non-membrane-proximal region.
  • the polypeptide GC3pep (membrane proximal end) and the polypeptide YP7pep (membrane proximal end) of human GPC3 were respectively coated on VHH 13A, 13B and Table 21, the S001-NB152-72-2 antibody can simultaneously bind the polypeptide GC3pep and the polypeptide YP7pep, and the S001-NB150-28 (cyno+) antibody can bind the polypeptide YP7pep , these two antibodies belong to the antibodies that recognize the near-membrane end epitope; the other antibodies do not bind the polypeptide GC3pep and the polypeptide YP7pep, and belong to the non-membrane end antibody.

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Abstract

提供了GPC3抗体及其应用，具体提供了特异性结合GPC3的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、分离的核酸片段、载体、宿主细胞、相应制备方法、药物组合物、治疗方法、制药用途、GPC3检测方法及检测试剂盒，在制备治疗癌症或肿瘤药物中具有重要意义。

Description

GPC3抗体及其应用
本公开要求于2020年12月10日提交中国专利局、申请号为202011437242.9、发明名称为“GPC3抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。本公开还要求于2021年11月16日提交中国专利局、申请号为202111353420.4、发明名称为“GPC3抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体领域，具体而言，涉及GPC3抗体及其应用。
背景技术
磷脂酰肌醇蛋白聚糖3(Glypican-3，GPC3)是一种硫酸乙酰肝素(HS)糖蛋白，属于硫酸类肝素蛋白聚糖家族成员，它通过磷脂酰肌醇(GPI)锚定在细胞膜表面。GPC3核心蛋白包括580个氨基酸，大小约70KD，它被弗林蛋白酶(Furin)剪切后，产生一个40kD的氨基(N)末端亚基和一个30kD的羧基(C)末端亚基，二者通过二硫键连接。GPC3的两条HS侧链结合在靠近C端的位置(Takahiro Nishida,Hiroaki Kataoka.Glypican 3-Targeted Therapy in Hepatocellular Carcinoma，Cancers 2019；11(9):1339)。
GPC3在胚胎中胚层组织的细胞增殖中发挥重要的调节作用，缺失GPC3基因会导致过度生长综合症，即Simpson-Golabi-Behmel综合症(SGBS)。GPC3在整个胎儿期均有明显表达，而出生后到成人阶段，除在胎盘、乳腺、间皮、卵巢、肺及肾组织有弱表达外，其他正常组织无明显表达。GPC3在成人多种肿瘤组织中异常表达，如肝细胞癌(HCC)、肺鳞癌、胃癌、卵巢癌等。尤其在HCC细胞中呈现高表达，它通过提高自分泌/旁分泌经典Wnt信号传递，促进HCC细胞的生长和侵袭(Capurro MI,Xiang Y-Y,Lobe C,Filmus J.Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling.Cancer Res 2005；65:6245–54.)。免疫组织化学染色检测发现大约70％的HCC病人肿瘤组织中呈现GPC3蛋白高表达(Capurro M,Wanless IR,Sherman M,et al.Glypican-3:a novel serum and histochemical marker for hepatocellular carcinoma.Gastroenterology 2003；125:89–97)，因此GPC3被认为是肿瘤治疗的一个候选靶标。
1993年比利时科学家Hamers-Casterman C在骆驼血液中发现一种只含重链不含轻链的天然重链抗体,重链抗体和普通的抗体相比虽然缺失了轻链,但是依然保留结合抗原的能力(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,et al.Naturally occurring antibodies devoid of light chains.Nature.363(6428):446–8(1993))。克隆骆驼体内重链抗体的可变区后,得到的仅由一个重链可变区组成的单域抗体(single domain antibody，sdAb)，称为纳米抗体或VHH结构域(variable heavy chain domain of a heavy chain antibody)。
与普通抗体相比，纳米抗体具有分子量小、稳定性好、可溶性高等诸多优点。因此应用纳米抗体技术研发GPC3抗体具有广阔的前景，获得GPC3特异性纳米抗体对相关治疗药物 或检测试剂的开发具有重要意义。
发明内容
本公开公开特异性结合GPC3的抗体或抗原结合片段、多肽、嵌合抗原受体、免疫效应细胞、分离的核酸片段、载体、宿主细胞、相应制备方法、药物组合物、治疗方法、制药用途、GPC3检测方法及检测试剂盒。
在第一方面，本公开涉及一种特异性结合GPC3的抗体或抗原结合片段，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3。
在一些具体的实施方式中，所述CDR1、CDR2和CDR3为HCDR1、HCDR2和HCDR3。
在一些具体的实施方式中，所述HCDR1、HCDR2和HCDR3根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定，例如选自表1；例如，所述HCDR1选自SEQ ID NO：24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、88、91或94，所述HCDR2选自SEQ ID：25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、89、92或95，和所述HCDR3选自SEQ ID：26、29、32、35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、90、93或96；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：24～26、27～29或30～32的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：33～35、36～38或39～41的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：42～44、45～47或48～50的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：51～53、54～56或57～59的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：60～62、63～65或66～68的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：69～71、72～74或75～77的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：78～80、81～83或84～86的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：88～90、91～93或94～96。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、 98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
在一些具体的实施方式中，所述单域抗体包含SEQ ID NO：17～23或87任一项所示序列；可选地，所述单域抗体包含与SEQ ID NO：17～23或87任一项所示序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或所述单域抗体包含与SEQ ID NO：17～23或87任一项所示序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述单域抗体包含SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区；可选地，所述单域抗体包含与SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列，或者所述单域抗体包含与SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些具体的实施方式中，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选的，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区，更优选具有如SEQ ID NO：1所示氨基酸序列；优选地，所述抗体或抗原结合片段为重链抗体。
在一些具体的实施方式中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在一些具体的实施方式中，所述VHH结构域特异性结合人、猴和/或鼠GPC3；优选地，其与人和/或猴GPC3结合的KD优于1.00E-7M、1.00E-8M、2.00E-8M、3.00E-8M、4.00E-8M、5.00E-8M、6.00E-8M、7.00E-8M、8.00E-8M、9.00E-8M、1.00E-9M、2.00E-9M、3.00E-9M、4.00E-9M、5.00E-9M、6.00E-9M、7.00E-9M、8.00E-9M、9.00E-9M或1.00E-10M。
在第二方面，本公开公开一种特异性结合GPC3的抗体或抗原结合片段，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3；所述CDR1、CDR2和CDR3包含选自SEQ ID NO:17～23或87任一项所示VHH结构域中的HCDR1、HCDR2和HCDR3。
在一些具体的实施方式中，所述HCDR1、HCDR2和HCDR3根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定，例如选自表1；例如，所述HCDR1选自SEQ ID NO：24、 27、30、33、36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、88、91或94，所述HCDR2选自SEQ ID：25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、89、92或95，和所述HCDR3选自SEQ ID：26、29、32、35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、90、93或96；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：24～26、27～29或30～32的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：33～35、36～38或39～41的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：42～44、45～47或48～50的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：51～53、54～56或57～59的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：60～62、63～65或66～68的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：69～71、72～74或75～77的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：78～80、81～83或84～86的序列；
优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：88～90、91～93或94～96。。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在第三方面，本公开还公开一种多肽，所述多肽包含前述抗体或抗原结合片段，优选地，所述多肽还连接有其他功能性分子，所述其他功能性分子可选自以下一种或多种：信号肽、蛋白标签、其他抗原结合分子或细胞因子。
在一些具体的实施方式中，所述其他抗原结合分子特异性结合GPC3以外的抗原或结合与前述抗体或抗原结合片段不同的GPC3表位；
优选地，所述GPC3以外的抗原可选自：CD3，优选CD3ε；CD16，优选CD16A；NKG2D；CD40；4-1BB；CD137或CD19；EGFR；EGFRvIII；mesothelin；HER2；EphA2；Her3；EpCAM；MUC1；MUC16；CEA；Claudin18.2；叶酸受体；Claudin6；WT1；NY-ESO-1；MAGE3；ASGPR1或CDH16；
优选地，所述其他抗原结合分子为抗体或抗原结合片段；
优选地，所述多肽为多特异性抗原结合分子，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性，更优选地，所述多特异性抗原结合分子可为二价、四价或六价。
在一些具体的实施方式中，所述细胞因子可选自IL2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha。
在第四方面，本公开还公开一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含前述抗体或抗原结合片段。
在第五方面，本公开还公开一种免疫效应细胞，所述免疫效应细胞表达前述的嵌合抗原受体，或包含编码前述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
在第六方面，本公开还公开一种分离的核酸片段，所述核酸片段编码前述的抗体或抗原结合片段、多肽、或嵌合抗原受体。
在第七方面，本公开还公开一种载体(vector)，所述载体包含前述的核酸片段。
在第八方面，本公开还公开一种宿主细胞，所述宿主细胞包含前述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在第九方面，本公开还公开一种制备前述抗体或抗原结合片段或多肽的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体或抗原结合片段，或分离所述细胞表达的多肽。
在第十方面，本公开还公开一种制备前述免疫效应细胞的方法，所述方法包括将编码前述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达前述CAR。
在第十一方面，本公开还公开一种药物组合物，所述药物组合物包含前述的抗体或抗原结合片段、多肽、免疫效应细胞、核酸片段、载体或根据前述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
在第十二方面，本公开还公开一种治疗GPC3阳性肿瘤或癌症的方法，所述方法包括向受试者施用有效量的前述的抗体或抗原结合片段、多肽、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或前述药物组合物；优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺癌。
在第十三方面，本公开还公开前述的抗体或抗原结合片段、多肽、免疫效应细胞、核酸 片段、载体、根据前述方法制备获得的产品或前述药物组合物在制备治疗GPC3阳性肿瘤或癌症药物中的用途，优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺癌。
在第十四方面，本公开还公开前述的抗体或抗原结合片段、多肽、免疫效应细胞、核酸片段、载体、根据前述方法制备获得对的产品或前述药物组合物，用于治疗GPC3阳性肿瘤或癌症；优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺癌。
在第十五方面，本公开还公开一种试剂盒，所述试剂盒包含前述的抗体或抗原结合片段、多肽、免疫效应细胞、核酸片段、载体、宿主细胞、根据前述方法制备获得的产品或前述药物组合物。
在第十六方面，本公开还公开一种检测生物学样品中GPC3表达的方法，所述方法包括在前述的抗体或抗原结合片段与GPC3之间能够形成复合物的条件下，使所述生物学样品与前述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中GPC3的存在或表达水平。
在第十七方面，本公开还公开前述抗体或抗原结合片段在制备GPC3检测试剂中的用途。
在第十八方面，本公开还公开一种用于检测GPC3的试剂盒，所述试剂盒至少包含前述的抗体或抗原结合片段。
术语定义和说明
除非本公开另外定义，与本公开相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“磷脂酰肌醇蛋白聚糖3(Glypican-3，GPC3)”是一种硫酸乙酰肝素(HS)糖蛋白，属于硫酸类肝素蛋白聚糖家族成员，它通过磷脂酰肌醇(GPI)锚定在细胞膜表面。本文“GPC3”包括成熟或未成熟的全长野生型GPC3蛋白或其突变体(例如点突变、插入突变或缺失突变)、剪切变体(splice variant)、直系同源物(Orthologs)以及前述GPC3的片段。示例性地，本文“GPC3”可以来源于哺乳动物，例如，人、灵长类动物，如猴(例如恒 河猴、食蟹猴)和啮齿类动物，例如小鼠和大鼠。示例性地，人GPC3氨基酸序列可参见NCBI:NM_004484.3，猴GPC3氨基酸序列可参见NCBI：XP_011739317.1。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10 -7M或更低、约10 -8M或更低、约1×10 -9M或更低、约1×10 -10M或更低、1×10 -11M或更低或1×10 -12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中的每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限 于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
本文术语“VHH结构域”和“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“单域抗体”、“VHH结构域”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。示例性地，本文“抗原结合片段”或“抗体片段”包括但不限于Fab、F(ab’) 2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠、小鼠、兔或羊驼)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985J Immunol Methods 125:191-202；以上通过援引加入并入本文。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系 统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
示例性地，采用Kabat、Chothia和IMGT确定S001-NB150-20(cyno+)、S001-NB150-28(cyno+)、S001-NB150-56、S001-NB152-1、S001-NB152-101、S001-NB152-72-2、VH001(SEQ ID NO：17～23)、VHH-12(SEQ ID NO：87)的CDR，具体结果如表1所示：
表1 CDR序列表
Figure PCTCN2021136637-appb-000001
Figure PCTCN2021136637-appb-000002
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”可选自CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文中的术语“Fc区”用于定义抗体重链中含有恒定区的至少一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。示例性地，人IgG重链Fc区可自Cys226或Pro230延伸至重链的羧基末端。然而，由宿主细胞生成的抗体可经历翻译后切割，自重链的C端切除一个或多个，特别是一个或两个氨基酸。因此，通过编码全长重链的特定核酸分子的表达由宿主细胞生成的抗体可包括全长重链，或者它可包括全长重链的切割变体。当重链的最终两个C端氨基酸是甘氨酸(G446)和赖氨酸(K447，编号方式依照Kabat EU索引)时可能就是这种情况。因此，Fc区的C端赖氨酸(Lys447)，或C端甘氨酸(Gly446)和赖氨酸(Lys447)可以存在或不存在。
典型地，IgG Fc区包含IgG CH2和IgG CH3域，可选地，在此基础上还可包含完整或部分铰链区，但不包含CH1域。人IgG Fc区的“CH2域”通常自约位置231处的氨基酸残基延伸至约位置340处的氨基酸残基。在一个实施方案中，碳水化合物链附着于CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中在CH2域C端的那段残基(即自IgG的约位置341处的氨基酸残基至约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域(例如具有在其一条链中引入的“隆起”(“节”，knob)和在其另一条链中相应引入的“空腔”(“穴”，hole)的CH3域；参见美国专利No.5,821,333，通过援引明确收入本文)。如本文中描述的，此类变体CH3域可用于促进两条不相同抗体重链的异二聚化。
除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号依照EU编号系统，也称作EU索引，如Kabat et al.,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,Bethesda,MD,1991中描述的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本公开所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本公开核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本公开蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位 BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本公开的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)， 无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本公开所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
本文术语“GPC3阳性”是指细胞或组织的GPC3细胞表达高于相应细胞或组织的正常表达水平，“GPC3阳性”可通过本领域已知方法检测。
附图说明
图1A为ELISA检测对照抗体与人GPC3-His蛋白的结合反应；
图1B为ELISA检测对照抗体与猴GPC3-His蛋白的结合反应；
图1C为ELISA检测对照抗体与鼠GPC3-His蛋白的结合反应；
图2A为ELISA检测多肽GC3pep蛋白与对照抗体的结合反应；
图2B为ELISA检测多肽YP7pep蛋白与对照抗体的结合反应；
图3A为Tab003抗体检测HepG2细胞GPC3表达量的FACS结果；
图3B为Tab005抗体检测HepG2细胞GPC3表达量的FACS结果；
图4A为Tab003抗体检测CHOK1-hGPC3.1C3细胞GPC3表达量的FACS结果；
图4B为Tab003抗体检测CHOK1-hGPC3.2B5细胞GPC3表达量的FACS结果；
图4C为为Tab003抗体检测CHOK1-hGPC3.3E9细胞GPC3表达量的FACS结果；
图5为Tab003抗体检测HEK293T-猴GPC3细胞GPC3表达量的FACS结果；
图6A为ELISA检测人GPC3蛋白免疫后羊驼血清抗体效价情况；
图6B为FACS检测人GPC3蛋白免疫后羊驼血清抗体效价情况；
图7A～图7B为ELISA检测VHH-hFc与人GPC3-his蛋白的结合反应；
图8A为FACS检测VHH-hFc与CHO-K1-人GPC3细胞的结合反应；
图8B为FACS检测VHH-hFc与CHO-K1细胞的结合反应；
图9A和图9C为FACS检测VHH-hFc与HepG2肿瘤细胞的结合反应；
图9B和图9D为FACS检测VHH-hFc与A431肿瘤细胞的结合反应；
图10为ELISA检测VHH-hFc与猴GPC3-His蛋白的结合反应；
图11为ELISA检测VHH-hFc与鼠GPC3-his蛋白的结合反应；
图12A为ELISA检测VHH-hFc与HEK293T-猴GPC3细胞的结合反应；
图12B为ELISA检测VHH-hFc与HEK293T细胞的结合反应；
图13A为ELISA检测VHH-hFc与GC3pep多肽蛋白的结合反应；
图13B为ELISA检测VHH-hFc与YP7pep多肽蛋白的结合反应。
具体实施方式
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
实施例1对照抗体制备、人源多肽制备、内源细胞鉴定和过表达细胞株的制备
(A)、对照抗体的制备
分别将识别人GPC3的单克隆抗体Y035和T2-23的VL和VH以及人IgG1Fc按照从N端到C端的顺序连接，其中VH和VL之间通过3个GGGGS连接子连接，形成scFv-人IgG1Fc(scFv-hFc)的形式，将其对应的核苷酸序列分别克隆到pTT5载体(购自优宝生物)中，并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。Y035和T2-23的的具体信息参见专利文献WO2017020812A1，其通过引用并入本文。将表达载体按照PEI(购自Polysciences)说明书瞬时转染HEK293E细胞(购自中国科学院典型培养物保藏委员会细胞库)并使用FreeStyle TM 293(Invitrogen)在37℃下连续培养5天，离心去除细胞成分，获得含ScFv-人IgG1 Fc(hFc)形式的抗体的培养上清液。将培养上清液上样到蛋白A层析柱(蛋白A填料AT Protein A Diamond和层析柱BXK16/26均购自博格隆)，使用PBS磷酸盐缓冲液(pH7.4)清洗后再用20mM PB,1M NaCl,pH 7.2进行清洗，最后使用pH3.4的柠檬酸缓冲液进行洗脱，收集从蛋白A层析柱上洗脱下来的带Fc标签的抗体，用1/10体积的pH8.0的1M Tris中和，用PBS在4℃条件透析过夜，透析后的抗体使用Nanodrop测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自安度斯)检测抗体内毒素含量，最后将抗体经0.22微米无菌过滤后分装于-80℃保存。将Y035scFv-hFc形式的抗体和T2-23scFv-hFc形式的抗体分别命名 为Tab003和Tab005。表2为相关对照抗体的序列信息。
表2对照抗体序列信息
Figure PCTCN2021136637-appb-000003
对照抗体与人GPC3-His蛋白(购自Acro，货号：GP3-H52H4)、猴GPC3-His蛋白(购自Acro，货号：GP3-C5225)以及鼠GPC3-His蛋白(购自Sino Biological，货号：50989-M08B)的结合活性如表3～5和图1A～1C所示，IgG亚型对照为人IgG1。结果说明，Tab003、Tab005抗体均与人GPC3蛋白以及猴GPC3蛋白有很好的结合活性，Tab003不与鼠GPC3蛋白结合，Tab005与鼠GPC3蛋白结合良好，与文献报道相符。
表3 ELISA检测对照抗体与人GPC3-his蛋白的结合反应
Figure PCTCN2021136637-appb-000004
表4 ELISA检测对照抗体与猴GPC3-his蛋白的结合反应
Figure PCTCN2021136637-appb-000005
表5 ELISA检测对照抗体与鼠GPC3-his蛋白的结合反应
Figure PCTCN2021136637-appb-000006
(B)、人源多肽GC3pep和YP7pep蛋白的制备
委托吉尔生化生产人源GPC3(NCBI:NM_004484.3，Ala524-Lys563)的多肽GC3pep和人源GPC3(NCBI:NM_004484.3，Asp511-Ser560)的多肽YP7pep，具体序列如下所示：
GC3pep：AELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLK(SEQ ID NO：8)；YP7pep：DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS(SEQ ID NO：9)。
对制备的上述多肽分别用识别不同表位的阳性对照抗体进行ELISA检测，检测结果如表6～7和图2A、2B所示，Tab005不能结合多肽GC3pep和YP7pep，Tab003能结合多肽GC3pep和YP7pep，检测结果与文献报道的一致，说明已经制备获得具有结合活性的上述多肽。
表6 ELISA检测对照抗体与多肽GC3pep蛋白的结合反应
Figure PCTCN2021136637-appb-000007
表7 ELISA检测对照抗体与多肽YP7pep蛋白的结合反应
Figure PCTCN2021136637-appb-000008
Figure PCTCN2021136637-appb-000009
(C)、内源性表达GPC3蛋白的细胞株鉴定
将HepG2细胞在T-75细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用Tab003和Tab005抗体作为一抗，FITC标记的二抗(购自Invitrogen，货号：A11013)经FACS(FACS CantoTM，购自BD公司)检测和分析。结果如表8以及图3A～3B所示，说明HepG2细胞与Tab003和Tab005均可结合。
表8内源细胞系HepG2细胞的FACS检测结果
Figure PCTCN2021136637-appb-000010
(D)、表达人GPC3蛋白的CHO-K1重组细胞株的制备
编码人GPC3全长氨基酸序列(NCBI:NM_004484.3)的核苷酸序列被克隆到pcDNA3.1载体(购自Clontech)并制备质粒。对CHO-K1细胞系(购自中国科学院典型培养物保藏委员会细胞库)进行质粒转染(
Figure PCTCN2021136637-appb-000011
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/mL puromycin的含10％(w/w)胎牛血清的DMEM/F12培养基中选择性培养2周，用Tab003抗体和驴抗人IgG H+L抗体(Jackson，货号：109605088)，在流式细胞仪(FACSAriaII，自BD Biosciences)上分选阳性单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存。具体选择结果如表9和图4A～4C所示，IgG亚型对照为人IgG1对照。表9说明，已经制得一系列人GPC3阳性表达的CHO-K1单克隆细胞系。图4A～4C中，横坐标为细胞荧光强度，纵坐标为细胞数。结果说明，1C3、2B5、3E9为人GPC3高水平表达细胞株，最终选择2B5细胞株用于后续实施例。
人GPC3全长氨基酸序列如下所示(SEQ ID NO：10)：
Figure PCTCN2021136637-appb-000012
表9表达人GPC3蛋白的CHO-K1重组细胞系FACS检测结果
Figure PCTCN2021136637-appb-000013
(E)、表达猴GPC3蛋白的重组HEK293T细胞株制备
编码猴GPC3全长氨基酸序列(NCBI：XP_011739317.1，SEQ ID NO：11)的核苷酸序列被克隆到pcDNA3.1载体(购自Thermofisher scientific)并制备质粒。对HEK293T细胞系用
Figure PCTCN2021136637-appb-000014
HD(Promega，货号：#E2311)进行质粒转染后，在含10μg/mL puromycin的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用Tab003抗体和和驴抗人IgG H+L抗体(Jackson，货号：109605088)在流式细胞仪(FACSAriaII，购自BD Biosciences)上分选富集阳性单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2培养，大约1周后进行扩增。对扩增后的细胞经流式细胞分析法进行检测，选择长势较好、荧光强度较高的细胞株继续扩大培养并液氮冻存。表达量的结果如图5，显示经过puromycin加压筛选后的HEK293T-猴-GPC3具有单一的阳性峰，可用于检测抗体的交叉活性。
猴GPC3全长氨基酸序列如下所示(SEQ ID NO：11)：
Figure PCTCN2021136637-appb-000015
实施例2针对GPC3的单域抗体VHH的制备
(A)、羊驼免疫及血清效价检测
委托吉尔生化生产偶联载体蛋白(KLH)的多肽GC3pep，即多肽GC3pep-KLH(AELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKC-KLH)。
采用人GPC3(Gln 25-His 559)-His蛋白(购自Acro，货号：GP3-H52H4)和多肽GC3pep-KLH，免疫两只羊驼(Alpaca，编号为NB150和NB152)。初次免疫时，人GPC3-His蛋白用弗氏完全佐剂乳化后皮下多点注射，即每只羊驼注射500μg人GPC3-His蛋白。前三次加强免疫时，人GPC3-His蛋白用弗氏不完全佐剂乳化后皮下多点注射，即每只羊驼注射250μg人GPC3-His蛋白。第四次加强免疫时，多肽GC3pep-KLH用弗氏不完全佐剂乳化后皮下多点注射，即每只羊驼注射250μg多肽GC3pep-KLH。初次免疫与第一次加强免疫之间 间隔3周，第一次加强免疫与第二次加强免疫之间间隔3周，第二次加强免疫与第三次加强免疫之间间隔3周，第三次加强免疫与第四次加强免疫之间间隔2个月。
每次加强免疫1周后采血，用ELISA和FACS检测血清中人GPC3-His的抗体效价和特异性，结果如图6A～6B和表10所示。说明经人GPC3-His和多肽GC3pep-KLH蛋白免疫后的羊驼的血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中ELISA最高稀释度在12500左右。其中空白对照为1％(w/w)BSA，批次指第四次(TB4)和第五次(TB5)免疫后第七天的羊驼血清，表中的数据为OD450nm值。
表10 ELISA检测人GPC3-His蛋白免疫后的羊驼血清抗体效价
Figure PCTCN2021136637-appb-000016
(B)、噬菌体文库构建及针对GPC3纳米抗体淘选
第五次(TB5)免疫后一周，采集100mL羊驼外周血，使用淋巴分离液分离PBMC，并使用RNAiso Plus试剂提取总RNA。使用PrimeScript TM II 1st Strand cDNA Synthesis Kit(购自Takara，货号：6210A)将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区核酸片段：
第一轮PCR：
上游引物(SEQ ID NO：12)：CTTGGTGGTCCTGGCTGC；
下游引物(SEQ ID NO：13)：GGTACGTGCTGTTGAACTGTTCC。
第二轮PCR：
以第一轮PCR产物作为模板，
上游引物(SEQ ID NO：14)：
Figure PCTCN2021136637-appb-000017
下游引物-1(SEQ ID NO：15)：
Figure PCTCN2021136637-appb-000018
下游引物-2(SEQ ID NO：16)：
Figure PCTCN2021136637-appb-000019
回收目标单域抗体核酸片段，并使用限制性内切酶SfiI将其克隆进入噬菌体展示用载体pcomb3XSS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对GPC3的单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为3.4×10 9。为检测文库的插入率，随机选取48个克隆做菌落PCR。结果显示插入率达到100％。
(C)、针对GPC3的单域抗体淘选
将人GPC3-Llama-Fc蛋白(购自Acro，货号：GP3-H5257)用pH值为9.6的碳酸盐缓冲液稀释至终浓度为5μg/mL，按100μL/孔加入酶标孔中，每个蛋白包被8孔，4℃包被过夜；弃包被液，PBS洗涤3次，每孔加入300μL 3％BSA-PBS封闭液，37℃封闭1小时；PBS洗涤3次，加入100μL噬菌体文库，37℃孵育1小时；吸出未结合的噬菌体，用PBST洗涤6次，PBS洗涤2次；加入100μL Gly-HCl洗脱液，37℃孵育8分钟，洗脱特异性结合的噬菌体；将该洗脱液转移至1.5mL无菌离心管中，迅速用10μL Tris-HCl中和缓冲液中和；取10μL进行梯度稀释，测定滴度，计算淘选回收率，其余洗脱物混合后进行扩增和纯化，可用于下一轮亲和淘选。
从第一轮淘选洗脱物滴度的平板上，用灭菌牙签随机挑取192个单克隆接种于1mL2×YT-A中，37℃，220r/min振荡培养8小时。取100μL上述培养物，按cell：phage＝1：20的比例加入M13K07噬菌体，37℃，静置15min，220r/min振荡培养45分钟。补加300μL体积的2×YT-AK，30℃，剧烈振荡培养过夜。第二天12000rpm离心2分钟，取上清，用于单克隆ELISA鉴定。
将人GPC3蛋白和GC3pep多肽用PH值为9.6的碳酸盐缓冲液分别稀释至终浓度为2μg/mL，按100μL孔加入酶标孔中，4℃包被过夜；弃包被液，PBST洗涤3次，每孔加入300μL5％脱脂牛奶，37℃封闭1小时；PBST洗涤3次，每孔加入50μL噬菌体培养菌液上清和50μL5％脱脂牛奶，37℃，孵育1小时；PBST洗涤5次，加入辣根过氧化物酶标记的抗M13抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1小时；PBST洗板6次。加入TMB显色液显色，100μL/孔，37℃，7分钟，加入终止液终止反应，50μL/孔，于450nm波长下测光密度。挑选人GPC3蛋白和GC3pep多肽双阳性克隆送成都擎科梓熙生物技术有限公司进行测序。
对测序结果进行分析，根据VHH编码蛋白序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列进行VHH-hFc生产鉴定。用于生产鉴定的VHH序列如下所示，并根据IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)确定所述VHH序列的CDR区，具体参见表1。
>S001-NB150-20(cyno+)(SEQ ID NO：17)
Figure PCTCN2021136637-appb-000020
>S001-NB150-28(cyno+)(SEQ ID NO：18)
Figure PCTCN2021136637-appb-000021
>S001-NB150-56(SEQ ID NO：19)
Figure PCTCN2021136637-appb-000022
>S001-NB152-1(SEQ ID NO：20)
Figure PCTCN2021136637-appb-000023
>S001-NB152-101(SEQ ID NO：21)
Figure PCTCN2021136637-appb-000024
>S001-NB152-72-2(SEQ ID NO：22)
Figure PCTCN2021136637-appb-000025
>VH001(SEQ ID NO：23)
Figure PCTCN2021136637-appb-000026
>VHH-12(SEQ ID NO:87)
Figure PCTCN2021136637-appb-000027
实施例3 VHH-hFc生产
将目标的VHH序列重组到人IgG1Fc的表达载体中，得重组质粒。具体的质粒构建、转染以及纯化流程参照实施例1(A)。将纯化的VHH-hFc进行蛋白浓度、纯度、内毒(Lonza试剂盒)等检测分析，结果如表11所示，结果发现，抗体最终产品纯度较高，内毒素浓度在1.0EU/mg以内。
表11纯化的VHH-hFc质量控制
Figure PCTCN2021136637-appb-000028
实施例4 VHH-hFc抗的鉴定
(A)、酶联免疫吸附实验(ELISA)检测VHH-hFc与人GPC3蛋白的结合
人GPC3蛋白用PBS稀释到终浓度1μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入100nM梯度稀释的VHH-hFc、阳性对照抗体或阴性对照抗体，50μl每孔。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记二抗(购自Merck，货号：AP113P)，37℃孵育1小时，用PBS洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值，VHH-hFc与人GPC3蛋白的结合活性结果如图7A～图7B和表12-1、表12-2所示，S001-NB152-1抗体与人GPC3蛋白有弱结合，其余7个VHH-hFc与人GPC3蛋白均结合良好。其中IgG对照为hIgG1，表中数据为OD450nm值。
表12-1 ELISA检测VHH-hFc与人GPC3蛋白的结合反应
Figure PCTCN2021136637-appb-000029
表12-2 ELISA检测VHH-hFc与人GPC3蛋白的结合反应
Figure PCTCN2021136637-appb-000030
(B)、流式细胞实验(FACS)检测抗体与表达GPC3蛋白的细胞和不表达GPC3蛋白的细胞(阴性细胞)的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，吸除培养基，用PBS缓冲液洗涤2次，用胰酶消化细胞，然后用完全培养基终止消化，并吹打细胞至单细胞悬液。细胞计 数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2x10 6细胞每毫升，按每孔50μl加入到96孔FACS反应板中，加入VHH-hFc、阳性对照抗体或阴性对照抗体，每孔50μl，4度孵育1小时。用PBS缓冲液离心洗涤3次，加入每孔50μl二抗anti-hIgG(H+L)Alexa 647(购自Jackson，货号：109-605-088)，冰上孵育1小时。用PBS缓冲液离心洗涤3次，100μl用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(FlowJo)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50。
分析结果如表13～14以及图8A～8B、图9A～9D所示(NB表示No binding，即不结合)，表13和图8A-8B表明VHH-hFc均可结合CHO-K1-人GPC3细胞，不结合CHO-K1细胞；表14-1、表14-2和图9A-9D表明VHH-hFc均可结合HepG2细胞，除抗体S001-NB150-20(cyno+)外，其余抗体均不结合A431细胞。
表13 FACS检测VHH-hFc与CHO-K1-人GPC3细胞和CHO-K1细胞结合反应
Figure PCTCN2021136637-appb-000031
表14-1 FACS检测VHH-hFc与HepG2细胞和A431细胞结合反应
Figure PCTCN2021136637-appb-000032
Figure PCTCN2021136637-appb-000033
表14-2 FACS检测VHH-hFc与HepG2细胞和A431细胞结合反应
Figure PCTCN2021136637-appb-000034
实施例5 VHH-hFc的交叉结合活性检测
(A)、ELISA检测VHH-hFc与猴GPC3蛋白和鼠GPC3蛋白的结合
将猴GPC3-His蛋白(购自Acro，货号：GP3-C5225)按照实施例4(A)的方法进行ELISA检测与数据分析。VHH-hFc与猴GPC3蛋白的ELISA结果如图10和表15所示，结果表明，S001-NB150-56抗体与猴GPC3蛋白有弱结合，S001-NB152-1抗体与猴GPC3蛋白不结合，其余VHH-hFc与猴GPC3蛋白结合良好。其中IgG对照为hIgG1，表中数据为OD450nm值。
表15 ELISA检测VHH-hFc与猴GPC3蛋白的结合反应
Figure PCTCN2021136637-appb-000035
将鼠GPC3-his蛋白(购自Sino Biological，货号：50989-M08B)按照实施例4(A)的方法进行ELISA检测与数据分析。VHH-hFc与鼠GPC3蛋白的ELISA结果如图11和表16所示，结果表明，S001-NB152-1抗体与鼠GPC3蛋白有弱结合，S001-NB150-20(cyno+)和S001-NB152-101与鼠GPC3蛋白不结合，其余4个VHH-hFc与鼠GPC3蛋白结合良好。
表16 ELISA检测VHH-hFc与鼠GPC3蛋白的结合反应
Figure PCTCN2021136637-appb-000036
(B)ELISA检测VHH-hFc与表达猴GPC3蛋白的细胞结合
将HEK293T-猴GPC3细胞按照实施例4(B)的方法进行FACS检测与数据分析。分析结果如表17以及图12A、图12B所示，所有VHH-hFc均与HEK293T-猴-GPC3细胞结合，与HEK293T细胞不结合。
表17 FACS检测VHH-hFc与HEK293T-猴GPC3细胞和HEK293T细胞结合反应
Figure PCTCN2021136637-appb-000037
实施例6 GPC3羊驼抗体的亲和力检测
(A)、VHH-hFc抗体与人GPC3蛋白亲和力检测
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗人GPC3 VHH-hFc。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的GPC3抗 原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的人GPC3-His持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1：1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1：1模型。VHH-hFc与人GPC3蛋白的结合速率(Ka)、解离速率(Kdis)及结合亲和力(KD)如表所示，其中抗体Tab003作为对照。如表18-1、表18-2和图13A和13B所示，VHH-hFc与人GPC3蛋白的亲和力均优于1E-7M，S001-NB150-28(cyno+)、S001-NB152-101和S001-NB152-72-2、VHH-12与人GPC3蛋白的亲和力接近1E-8M，其余VHH-hFc与人GPC3蛋白的亲和力均优于1E-8M，VH001的亲和力接近1E-9M。
表18-1 VHH-hFc与人GPC3蛋白的结合亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S001-NB150-20(cyno+) 3.98E+05 2.41E-03 6.06E-09
S001-NB150-28(cyno+) 2.97E+05 5.74E-03 1.93E-08
S001-NB150-56 1.04E+05 7.57E-04 7.25E-09
S001-NB152-1 1.53E+05 1.14E-03 7.45E-09
S001-NB152-101 1.51E+05 1.67E-03 1.11E-08
S001-NB152-72-2 1.53E+05 1.78E-03 1.16E-08
VH001 2.67E+05 4.43E-04 1.66E-09
Tab003 2.62E+05 3.58E-04 1.36E-09
表18-2 VHH-hFc与人GPC3蛋白的结合亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
VHH-12 1.44E+05 2.40E-03 1.67E-08
Tab003 1.22E+05 4.26E-04 3.51E-09
Tab005 2.08E+05 4.42E-05 2.13E-10
(B)、GPC3羊驼抗体与猴GPC3-his蛋白亲和力检测
按照实施例6(A)的方法对VHH-hFc与猴GPC3-His蛋白进行亲和力检测，其中抗体Tab003作为对照。如表19所示，VHH-hFc与猴GPC3蛋白的亲和力优于1E-8M。
表19 VHH-hFc与猴GPC3蛋白的结合亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S001-NB150-20(cyno+) 2.43E+05 1.03E-03 4.23E-09
S001-NB150-28(cyno+) 3.06E+05 1.57E-03 5.12E-09
S001-NB150-56 2.36E+05 1.09E-03 4.64E-09
S001-NB152-1 1.75E+05 9.83E-04 5.63E-09
S001-NB152-101 1.81E+05 4.80E-04 2.66E-09
S001-NB152-72-2 2.34E+05 7.54E-04 3.23E-09
VH001 3.52E+05 1.08E-03 3.06E-09
Tab003 2.42E+05 3.09E-04 1.28E-09
(C)、GPC3羊驼抗体与鼠GPC3-his蛋白亲和力检测
按照实施例6(A)的方法对VHH-hFc与鼠GPC3-His蛋白进行亲和力检测，其中抗体Tab005作为对照。如表20所示，S001-NB150-20(cyno+)和S001-NB152-101与鼠GPC3蛋白不结合，其余5个VHH-hFc与鼠GPC3蛋白的亲和力均优于1E-7M，其中VH001与鼠GPC3蛋白结合的亲和力在1E-8M左右，S001-NB150-28(cyno+)、S001-NB150-56、S001-NB152-1和S001-NB152-72-2的亲和力则均在1E-8M以上。
表20 VHH-hFc与鼠GPC3蛋白的结合亲和力
Figure PCTCN2021136637-appb-000038
实施例7抗体抗原结合表位(epitope)分析
(A)抗体抗原结合区域的鉴定
成熟的GPC3蛋白具有一个40kD左右可溶性的能够进入血液的的氨基端(N末端)肽和一个30kD左右的膜结合的羧基端(C末端)肽。Tab003抗体识别GPC3蛋白C末端靠近细胞膜的区域(近膜端)，Tab005抗体识别非近膜端区域。为了鉴定VHH-hFc的抗原结合表位是否位于近膜端，按照实施例4(A)ELISA方法，分别包被人源GPC3的多肽GC3pep(近膜端)和多肽YP7pep(近膜端)对VHH-hFc进行近膜端结合鉴定，如图13A、13B和表21所示，S001-NB152-72-2抗体可同时结合多肽GC3pep和多肽YP7pep，S001-NB150-28(cyno+)抗体可结合多肽YP7pep，这两种抗体都属于识别近膜端表位的抗体；其余抗体均不结合多肽GC3pep和多肽YP7pep，属于非近膜端抗体。
表21 ELISA方法对VHH-hFc进行近膜端和非近膜端表位分类
Figure PCTCN2021136637-appb-000039
Figure PCTCN2021136637-appb-000040

Claims (28)

  1. 一种特异性结合GPC3的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3；所述CDR1、CDR2和CDR3包含选自SEQ ID NO:17～23或87任一项所示VHH结构域中的HCDR1、HCDR2和HCDR3。
  2. 根据权利要求1所述的抗体或抗原结合片段，其中，所述HCDR1、HCDR2和HCDR3根据IMGT编号系统、Kabat编号系统或Chothia编号系统确定，例如选自表1；例如，所述HCDR1选自SEQ ID NO：24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、88、91或94，所述HCDR2选自SEQ ID：25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、85、89、92或95，和所述HCDR3选自SEQ ID：26、29、32、35、38、41、44、47、50、53、56、59、62、65、68、71、74、77、80、83、86、90、93或96；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：24～26、27～29或30～32的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：33～35、36～38或39～41的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：42～44、45～47或48～50的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：51～53、54～56或57～59的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：60～62、63～65或66～68的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：69～71、72～74或75～77的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：78～80、81～83或84～86的序列；
    优选地，根据IMGT编号系统、Kabat编号系统或Chothia编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：88～90、91～93或94～96。
  3. 根据权利要求1或2所述的抗体或抗原结合片段，其中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  4. 根据权利要求1或2所述的抗体或抗原结合片段，其中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  5. 根据权利要求1～4任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
  6. 根据权利要求1～5任一项所述的抗体或抗原结合片段，其中，所述单域抗体包含SEQ  ID NO：17～23或87任一项所示序列；可选地，所述单域抗体包含与SEQ ID NO：17～23或87任一项所示序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或所述单域抗体包含与SEQ ID NO：17～23或87任一项所示序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  7. 根据权利要求1～6任一项所述的抗体或抗原结合片段，其中，所述单域抗体包含SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区；可选地，所述单域抗体包含与SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列，或者所述单域抗体包含与SEQ ID NO：17～23或87任一项所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  8. 根据权利要求1～7任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  9. 根据权利要求1～8任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区，更优选具有如SEQ ID NO：1所示氨基酸序列；优选地，所述抗体或抗原结合片段为重链抗体。
  10. 根据权利要求1～9任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  11. 根据权利要求1～10任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段特异性结合人、猴和/或鼠GPC3；优选地，其与人、猴和/或鼠GPC3结合的KD优于1.00E-7M、1.00E-8M、2.00E-8M、3.00E-8M、4.00E-8M、5.00E-8M、6.00E-8M、7.00E-8M、8.00E-8M、9.00E-8M、1.00E-9M、2.00E-9M、3.00E-9M、4.00E-9M、5.00E-9M、6.00E-9M、7.00E-9M、8.00E-9M、9.00E-9M或1.00E-10M。
  12. 一种多肽，其中，所述多肽包含权利要求1～11任一项所述抗体或抗原结合片段，优选地，所述多肽还连接有其他功能性分子，优选地，所述其他功能性分子选自以下一种或多种：信号肽、蛋白标签、其他抗原结合分子或细胞因子。
  13. 根据权利要求12所述的多肽，其中，所述其他抗原结合分子特异性结合GPC3以外的抗原或结合与权利要求1～11任一项所述抗体或抗原结合片段不同的GPC3表位；
    优选地，所述GPC3以外的抗原选自：CD3，优选CD3ε；CD16，优选CD16A；NKG2D；CD40；4-1BB；CD137或CD19；EGFR；EGFRvIII；mesothelin；HER2；EphA2；Her3；EpCAM； MUC1；MUC16；CEA；Claudin18.2；叶酸受体；Claudin6；WT1；NY-ESO-1；MAGE3；ASGPR1或CDH16；
    优选地，所述其他抗原结合分子为抗体或抗原结合片段；
    优选地，所述多肽为多特异性抗原结合分子，所述多特异性抗原结合分子为双特异性、三特异性或四特异性，更优选地，所述多特异性抗原结合分子为二价、四价或六价。
  14. 根据权利要求12所述的多肽，其中，所述细胞因子选自IL2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha。
  15. 一种嵌合抗原受体(CAR)，其中，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1～11任一项所述抗体或抗原结合片段。
  16. 一种免疫效应细胞，其中，所述免疫效应细胞表达权利要求15所述的嵌合抗原受体，或包含编码权利要求15所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  17. 一种分离的核酸片段，其中，所述核酸片段编码权利要求1～11任一项所述抗体或抗原结合片段，或权利要求12～14任一项所述多肽，或权利要求15所述嵌合抗原受体。
  18. 一种载体(vector)，其中，所述载体包含权利要求17所述的核酸片段。
  19. 一种宿主细胞，其中，所述宿主细胞包含权利要求18所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
  20. 一种制备权利要求1～11任一项所述抗体或抗原结合片段或权利要求12～14任一项所述多肽的方法，其中，所述方法包括培养权利要求19所述细胞，以及分离所述细胞表达的抗体或抗原结合片段，或分离所述细胞表达的多肽。
  21. 一种制备权利要求16所述免疫效应细胞的方法，其中，所述方法包括将编码权利要求15所述的嵌合抗原受体的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求15所述的嵌合抗原受体。
  22. 一种药物组合物，其中，所述药物组合物包含权利要求1～11任一项所述的抗体或抗原结合片段、权利要求12～14任一项所述的多肽、权利要求16所述免疫效应细胞、权利要求17所述的核酸片段、权利要求18所述载体或根据权利要求20-21任一项所述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
  23. 一种治疗GPC3阳性肿瘤或癌症的方法，其中，所述方法包括向受试者施用有效量的权利要求1～11任一项所述的抗体或抗原结合片段、权利要求12～14任一项所述的多肽、权利要求16所述免疫效应细胞、权利要求17所述的核酸片段、权利要求18所述载体、根据权利要求20-21任一项所述方法制备获得的产品或权利要求22所述的药物组合物；优选地，所述 GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺癌。
  24. 权利要求1～11任一项所述的抗体或抗原结合片段、权利要求12～14任一项所述的多肽、权利要求16所述免疫效应细胞、权利要求17所述的核酸片段、权利要求18所述载体、根据权利要求20-21任一项所述方法制备获得的产品或权利要求22所述的药物组合物在制备治疗GPC3阳性肿瘤或癌症药物中的用途；优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺。
  25. 权利要求1～11任一项所述的抗体或抗原结合片段、权利要求12～14任一项所述的多肽、权利要求16所述免疫效应细胞、权利要求17所述的核酸片段、权利要求18所述载体、根据权利要求20-21任一项所述方法制备获得的产品或权利要求22所述的药物组合物，其中，用于治疗GPC3阳性肿瘤或癌症；优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌、乳腺癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤或食道癌；更优选地，所述GPC3阳性肿瘤或癌症选自肝癌、胃癌、肺癌或乳腺。
  26. 一种试剂盒，其中，所述试剂盒包含权利要求1～11任一项所述的抗体或抗原结合片段、权利要求12～14任一项所述的多肽、权利要求16所述免疫效应细胞、权利要求17所述的核酸片段、权利要求18所述载体、权利要求19所述宿主细胞、根据权利要求20-21任一项所述方法制备获得的产品或权利要求22所述药物组合物。
  27. 一种检测生物学样品中GPC3表达的方法，其中，所述方法包括在权利要求1～11任一项所述的抗体或抗原结合片段与GPC3之间能够形成复合物的条件下，使所述生物学样品与权利要求1～11任一项所述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中GPC3的存在或表达水平。
  28. 权利要求1～11任一项所述抗体或抗原结合片段在制备GPC3检测试剂中的用途。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023246574A1 (zh) * 2022-06-24 2023-12-28 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途
WO2024114410A1 (zh) * 2022-11-28 2024-06-06 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022739A1 (ja) * 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha Gpc3の血中可溶化ｎ端ペプチドまたはｃ端ペプチドに対する抗体
CN108659129A (zh) * 2018-05-17 2018-10-16 新疆大学 一种抗gpc3蛋白的纳米抗体及其制备方法和应用
CN110872351A (zh) * 2019-09-06 2020-03-10 广西科技大学 一种特异性结合gpc3蛋白的纳米抗体gn1及其制备方法和应用
CN111320694A (zh) * 2020-02-15 2020-06-23 广西科技大学 一种由重链抗体的可变区组成的纳米抗体gn2及其制备方法和应用
CN111732659A (zh) * 2020-07-21 2020-10-02 珠海中科先进技术研究院有限公司 一种磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法与应用
CN111848802A (zh) * 2020-07-21 2020-10-30 珠海中科先进技术研究院有限公司 一种具有突出热稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法
CN111848803A (zh) * 2020-07-21 2020-10-30 珠海中科先进技术研究院有限公司 一种具有突出酸碱稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法
CN111909274A (zh) * 2020-07-21 2020-11-10 珠海中科先进技术研究院有限公司 一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022739A1 (ja) * 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha Gpc3の血中可溶化ｎ端ペプチドまたはｃ端ペプチドに対する抗体
CN108659129A (zh) * 2018-05-17 2018-10-16 新疆大学 一种抗gpc3蛋白的纳米抗体及其制备方法和应用
CN110872351A (zh) * 2019-09-06 2020-03-10 广西科技大学 一种特异性结合gpc3蛋白的纳米抗体gn1及其制备方法和应用
CN111320694A (zh) * 2020-02-15 2020-06-23 广西科技大学 一种由重链抗体的可变区组成的纳米抗体gn2及其制备方法和应用
CN111732659A (zh) * 2020-07-21 2020-10-02 珠海中科先进技术研究院有限公司 一种磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法与应用
CN111848802A (zh) * 2020-07-21 2020-10-30 珠海中科先进技术研究院有限公司 一种具有突出热稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法
CN111848803A (zh) * 2020-07-21 2020-10-30 珠海中科先进技术研究院有限公司 一种具有突出酸碱稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法
CN111909274A (zh) * 2020-07-21 2020-11-10 珠海中科先进技术研究院有限公司 一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANG JIA-HAO, FU-MOU SUN, YU HAN, YA WANG, CAI JIA-LING, MIN WANG, JUAN ZHANG: "Screening and characterization of human anti-GPC3 single chain Fv antibody fragment selected by phage display", ACTA PHARMACEUTICA SINICA, YAOXUE XUEBAO, CN, vol. 52, 31 December 2017 (2017-12-31), CN , pages 1877 - 1883, XP055943367, ISSN: 0513-4870, DOI: 10.16438/j.0513-4870.2017-0687 *
XIA LIJIE, TENG QIAO, CHEN QI, ZHANG FUCHUN: "Preparation and Characterization of Anti-GPC3 Nanobody Against Hepatocellular Carcinoma", INTERNATIONAL JOURNAL OF NANOMEDICINE, vol. Volume 15, pages 2197 - 2205, XP055943366, DOI: 10.2147/IJN.S235058 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023246574A1 (zh) * 2022-06-24 2023-12-28 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途
WO2024114410A1 (zh) * 2022-11-28 2024-06-06 四川科伦博泰生物医药股份有限公司 靶向gpc3的抗体及其用途

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