WO2021110095A1 - 抗gpc3抗体、其抗原结合片段及其医药用途 - Google Patents

抗gpc3抗体、其抗原结合片段及其医药用途 Download PDF

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WO2021110095A1
WO2021110095A1 PCT/CN2020/133588 CN2020133588W WO2021110095A1 WO 2021110095 A1 WO2021110095 A1 WO 2021110095A1 CN 2020133588 W CN2020133588 W CN 2020133588W WO 2021110095 A1 WO2021110095 A1 WO 2021110095A1
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seq
heavy chain
variable region
antibody
light chain
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PCT/CN2020/133588
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English (en)
French (fr)
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花海清
余华星
何娟梅
包如迪
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上海翰森生物医药科技有限公司
江苏豪森药业集团有限公司
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Priority to CN202080006983.3A priority Critical patent/CN113227148B/zh
Publication of WO2021110095A1 publication Critical patent/WO2021110095A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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

Definitions

  • This application relates to an anti-GPC3 antibody that is specifically immunoreactive to human GPC3, and antigen-binding fragments thereof, a chimeric antibody containing the CDR region of the anti-GPC3 antibody, a humanized antibody, and a human anti-GPC3 antibody
  • Glypican 3 (Glypican 3, GPC3) is a membrane protein of about 70kd, belonging to the Glypicans family, as an extracellular matrix protein in organ formation, which plays a role in cell adhesion or as a receptor for cell growth factors. effect. After GPC3 is expressed, it will be cleaved by furin enzyme to produce a soluble part of about 40kDa and a part of about 30kDa at the N-terminal, which is anchored to the C-terminal of the cell membrane by GPI molecules.
  • GPC3 is expressed in embryonic tissues (especially liver and kidney) and is an extracellular matrix protein related to organ formation. In adult tissues, GPC3 expression is not observed outside the placenta, but expression is observed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. It can be seen that, like alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and other proteins, GPC3 is also a protein expressed in embryonic tissues, so it is classified as an embryonic cancer antigen. That is, GPC3 is characterized in that it is not expressed in normal tissue cells but specifically expressed in cancer cells, so it can be used as a target molecule and tumor marker for cancer therapy.
  • AFP alpha-fetoprotein
  • CEA carcinoembryonic antigen
  • GPC3 is also a protein expressed in embryonic tissues, so it is classified as an embryonic cancer antigen. That is, GPC3 is characterized in that it is not expressed
  • GPC3 plays an important role in maintaining the activation of the Wnt pathway and the Hedgehogs pathway.
  • heparan sulfate molecules coupled with GPC3 can enhance the binding of Wnts to their receptors, which plays an important role in maintaining the Wnt pathway.
  • GPC3 is expressed in the brain, digestive tract, bladder, gonads and skin, and is highly expressed on the surface of hepatocellular carcinoma; Wnt pathway plays an important role in the development of liver cancer, such as 20% of hepatocellular carcinoma ⁇ -Catenin pathway mutation and Frizzled-7 receptor The body is overexpressed, so GPC3 may play a role in promoting the occurrence of some hepatocellular carcinomas.
  • Codrituzumab (coltrastuzumab; alias GC33) developed by Roche, patent number: US20100248359, this antibody drug targets the C-terminal 524 of human GPC3 -563 amino acids, belonging to human IgG1, kappa subtype.
  • GC33 can induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and has a significant inhibitory effect on tumor growth in the HuH-7 tumor pharmacodynamic mouse model.
  • GC33 is in clinical phase II (NCT01507168), and its indications include hepatocellular carcinoma (HCC), metastatic hepatocellular carcinoma, liver cancer, metastatic liver cancer, etc.
  • HCC hepatocellular carcinoma
  • metastatic liver cancer metastatic liver cancer
  • the stability of the antibody is closely related to the drug effect, metabolism and safety in the human body. For example, the better the stability of the antibody, the less immunogenicity it produces.
  • the application provides an anti-GPC3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and/or a light chain variable region selected from:
  • the heavy chain variable region includes at least one HCDR1 selected from the following sequences: SEQ ID NO: 7, SEQ ID NO: 8,
  • the antibody heavy chain variable region includes at least one HCDR2 selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
  • the antibody heavy chain variable region includes at least one HCDR3 selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; and
  • the antibody light chain variable region includes at least one LCDR1 selected from the following sequence: SEQ ID NO: 15, SEQ ID NO: 16,
  • antibody light chain variable region includes at least one LCDR2 selected from the following sequence: SEQ ID NO: 17, SEQ ID NO: 18,
  • antibody light chain variable region includes at least one LCDR3 selected from the following sequence: SEQ ID NO: 19, SEQ ID NO: 20.
  • the CDR sequence of the antibody or antigen-binding fragment thereof may have a mutation of 1-3 amino acids, wherein a mutation of 1-3 amino acids refers to the insertion of any 1-3 amino acids , Deletion or substitution; specifically, the 1-3 amino acid mutation is an insertion, deletion or substitution of 1-3 amino acid that can optimize antibody activity, antibody stability, or reduce immunogenicity.
  • HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12 respectively;
  • HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13, respectively.
  • HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 14, respectively.
  • LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 respectively;
  • LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, respectively.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises : HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12 respectively; the light chain variable region includes: SEQ ID NO: 15 SEQ ID NO: 17 respectively And the LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 19.
  • This application also relates to a specific solution, an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes:
  • variable region of the light chain comprises:
  • SEQ ID NO: 15 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 15
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13, respectively;
  • the light chain variable region includes: LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, respectively.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes:
  • the antibody light chain variable region comprises:
  • SEQ ID NO: 15 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 15
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes:
  • HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 14 respectively;
  • the light chain variable region includes: LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, respectively.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: SEQ ID NO: 8 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 8 or SEQ ID NO: 11 or 1 compared with SEQ ID NO: 11. -HCDR2 with 3 amino acid mutations and SEQ ID NO: 14 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 14;
  • the light chain variable region includes: SEQ ID NO: 16 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 16 or SEQ ID NO: 18 or 1 compared with SEQ ID NO: 18. -LCDR2 with 3 amino acid mutations and SEQ ID NO: 20 or LCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 20.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12, respectively; the light chain variable region includes: SEQ ID NO: 16 SEQ ID NO: 18 and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 20.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: SEQ ID NO: 7 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 7 or SEQ ID NO: 9 or 1 compared with SEQ ID NO: 9 -HCDR2 with 3 amino acid mutations and SEQ ID NO: 12 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 12; the light chain variable region includes: SEQ ID NO: 16 or SEQ ID NO: 16 ID NO: 16 LCDR1 with 1-3 amino acid mutations compared to SEQ ID NO: 18 or LCDR2 with 1-3 amino acid mutations compared to SEQ ID NO: 18 and SEQ ID NO: 20 or with SEQ ID NO : 20 compared to LCDR3 with 1-3 amino acid mutations.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13, respectively; the light chain variable region includes: The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes SEQ ID NO: 7 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 7 or SEQ ID NO: 10 or 1 compared with SEQ ID NO: 10.
  • the light chain variable region includes: SEQ ID NO: 16 or SEQ ID NO: 16 ID NO: 16 LCDR1 with 1-3 amino acid mutations compared to SEQ ID NO: 18 or LCDR2 with 1-3 amino acid mutations compared to SEQ ID NO: 18 and SEQ ID NO: 20 or with SEQ ID NO : 20 compared to LCDR3 with 1-3 amino acid mutations.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 14, respectively; the light chain variable region includes: The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes: SEQ ID NO: 8 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 8 or SEQ ID NO: 11 or 1 compared with SEQ ID NO: 11. -HCDR2 with 3 amino acid mutations and SEQ ID NO: 14 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 14; the light chain variable region includes: SEQ ID NO: 15 or SEQ ID NO: 15 ID NO: 15 compared to LCDR1 with 1-3 amino acid mutations, SEQ ID NO: 17 or LCDR2 with 1-3 amino acid mutations compared to SEQ ID NO: 17, and SEQ ID NO: 19 or with SEQ ID NO : 19 compared to LCDR3 with 1-3 amino acid mutations.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the antibody is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a human antibody or a fragment thereof And humanized antibodies or fragments thereof.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a weight derived from human IgG1, IgG2, IgG3 or IgG4 Chain constant region or variants thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4.
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1.
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 41.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a mutated IgG1 heavy chain constant region, which is compared with The parent amino acid has enhanced ADCC toxicity.
  • the mutated IgG1 heavy chain constant region is shown in SEQ ID NO: 40.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant derived from a human kappa chain and a lambda chain. Region or its mutants;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain;
  • the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:42.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences: SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26.
  • This application also relates to a preferred solution, an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof contains at least 70%, 75%, 80% compared with the following sequence: %, 85%, 90%, 95% or 99% identical heavy chain variable regions: SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26.
  • reference to “at least 70% identity” means, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, 100% identity, or the range between any two values.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences: SEQ ID NO: 22 or SEQ ID NO: 25.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical light chain variable regions: SEQ ID NO: 22 or SEQ ID NO: 25.
  • the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 22;
  • the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 25;
  • the heavy chain variable region is SEQ ID NO: 23 and the light chain variable region is SEQ ID NO: 22;
  • the heavy chain variable region is SEQ ID NO: 23 and the light chain variable region is SEQ ID NO: 25;
  • the heavy chain variable region is SEQ ID NO: 24 and the light chain variable region is SEQ ID NO: 22;
  • the heavy chain variable region is SEQ ID NO: 24 and the light chain variable region is SEQ ID NO: 25;
  • variable region of the heavy chain is SEQ ID NO: 26 and the variable region of the light chain is SEQ ID NO: 22; or
  • the heavy chain variable region is SEQ ID NO: 26 and the light chain variable region is SEQ ID NO: 25.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 80%, 85% compared with the following sequence, 90%, 95%, 99%, or 100% identical heavy chain: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO : 37, SEQ ID NO: 38 or SEQ ID NO: 39.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 80%, 85% compared with the following sequence, 90%, 95%, 99%, or 100% identical light chain: SEQ ID NO: 28 or SEQ ID NO: 31.
  • the heavy chain shown in SEQ ID NO: 32 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 32 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity.
  • the heavy chain is SEQ ID NO: 27 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 27 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 29 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 29 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 32 and the light chain is SEQ ID NO: 28; or
  • the heavy chain is SEQ ID NO: 32 and the light chain is SEQ ID NO: 31.
  • the heavy chain shown in SEQ ID NO: 36 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 36 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 37 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 37 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 38 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 38 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 39 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95% or 99% identity; or,
  • the heavy chain shown in SEQ ID NO: 39 or a heavy chain with at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity.
  • This application also relates to some embodiments, providing an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody comprises:
  • the heavy chain is SEQ ID NO: 36 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 36 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 37 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 37 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 38 and the light chain is SEQ ID NO: 28;
  • the heavy chain is SEQ ID NO: 38 and the light chain is SEQ ID NO: 31;
  • the heavy chain is SEQ ID NO: 39 and the light chain is SEQ ID NO: 28; or
  • the heavy chain is SEQ ID NO: 39 and the light chain is SEQ ID NO: 31.
  • the application further provides a polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof as described above.
  • the application further provides an expression vector containing the polynucleotide as described above.
  • the application further provides a host cell which is introduced or contains the expression vector as described above.
  • a host cell as described above wherein the host cell is bacteria, specifically Escherichia coli.
  • a host cell as described above wherein the host cell is a mammalian cell, specifically a CHO cell or a HEK293 cell.
  • This application further provides a method for producing an anti-GPC3 antibody or an antigen-binding fragment thereof, comprising: culturing the above-mentioned host cell (for example, HEK293 cell); separating the antibody from the culture (for example, separating the antibody from the cell culture fluid); and The antibody is purified (for example, the antibody is purified by chromatography).
  • a host cell for example, HEK293 cell
  • separating the antibody from the culture for example, separating the antibody from the cell culture fluid
  • the antibody is purified (for example, the antibody is purified by chromatography).
  • the application further provides a pharmaceutical composition, which contains the aforementioned anti-GPC3 antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, diluent or carrier.
  • a detection or diagnosis kit which contains the anti-GPC3 antibody or antigen-binding fragment thereof described in the present application, and one or more types capable of detecting the anti-GPC3 antibody Or a reagent that binds to GPC3 (or its epitope) by its antigen-binding fragment.
  • a detection or diagnosis kit which contains the anti-GPC3 antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present application, and can be used for detection or Diagnostic labeled secondary antibody, buffer and substrate.
  • an anti-GPC3 antibody or antigen-binding fragment thereof as described above or a composition as described above is used in the preparation of a medicament for the treatment or prevention of GPC3-mediated diseases or disorders use.
  • an anti-GPC3 antibody or antigen-binding fragment thereof or the composition as described above in the preparation of reagents or kits, wherein the reagents or kits can be used For the detection, diagnosis, and prognosis of diseases or conditions mediated by GPC3.
  • an anti-GPC3 antibody or antigen-binding fragment thereof or a composition as described above is used for the detection, diagnosis, and prognosis of GPC3-mediated diseases.
  • the application further provides a method for treating or preventing GPC3-mediated diseases, including the steps of: providing a subject with a therapeutically effective amount or a preventively effective amount of the anti-GPC3 antibody or antigen-binding fragment thereof as described above; or The subject provides a therapeutically effective amount or a prophylactically effective amount of the aforementioned pharmaceutical composition.
  • the disease or disorder described in this application is cancer; specifically, the disease or disorder is GPC3-mediated cancer; further, the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney Cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma or melanoma.
  • This application provides an anti-GPC3 antibody or antigen-binding fragment, which can specifically bind to GPC3 antigen (or its epitope) and cells expressing GPC3, has significant CDC activity, and has a significant killing effect on tumors.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present application has good endocytosis and is suitable for coupling with drugs.
  • the anti-GPC3 antibody or antigen-binding fragment of the present application has lower immunogenicity and higher stability, and is significantly better than Codrituzumab.
  • the anti-GPC3 antibody or antigen-binding fragment of the present application has better potential as an anti-cancer drug and ensures the safety of medication.
  • Figure 1 Humanized antibody Ab9 heavy chain variable region, GC33 antibody heavy chain variable region and its human germline template sequence correspondence (Note: the gray shading indicates the amino acid that is inconsistent with the corresponding human germline template) .
  • Figure 2 Humanized antibody Ab9 light chain variable region, GC33 antibody light chain variable region and its human germline template sequence correspondence (Note: the gray shading marks the amino acids that are inconsistent with the corresponding human germline template) .
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into kappa chain or lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain described in this application may further comprise a light chain constant region derived from human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain described in this application may further comprise a heavy chain constant region derived from human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment described in this application comply with the known Kabat or Chothia or ABM definition rules.
  • the amino acid numbering of the variable region adopts the Kabat numbering system.
  • Those skilled in the art can determine the corresponding relationship of a specific amino acid position in different numbering systems based on common sense.
  • GPC3 includes any variant or isoform of GPC3 that is naturally expressed by the cell.
  • the antibody (or antigen-binding fragment thereof) of the present application can cross-react with GPC3 of non-human species.
  • the antibody may also be specific for human GPC3, and may not show cross-reactivity with other species.
  • GPC3 or any variants or isoforms thereof can be isolated from cells or tissues, or produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • the anti-GPC3 antibody targets human GPC3 (or its epitope) with a normal glycosylation pattern.
  • murine antibody in this application refers to a monoclonal antibody against human GPC3 (or its epitope) prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with GPC3 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine GPC3 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 and IgG2. , IgG3 or IgG4 or its variant heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present application may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” does not include “humanized antibodies”.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region.
  • Humanized antibodies help overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry non-human protein components.
  • the variable region of the humanized antibody can be subjected to minimal back mutation to maintain the activity.
  • chimeric antibody is an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can reduce the immune response induced by the antibody of the first species.
  • a hybridoma secreting the monoclonal antibody of the first species is now established, and then the variable region genes are cloned from the hybridoma cells, and then the constant region genes of the second species antibody are cloned as needed.
  • the variable region gene and the constant region gene are connected to form a chimeric gene and then inserted into the vector, and finally the chimeric antibody molecule is expressed in an industrial system.
  • the constant region may be selected from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 of the second species or variants thereof; specifically, it comprises the heavy chain constant region of IgG1, IgG2 or IgG4 of the second species, or has enhanced ADCC toxicity IgG1 heavy chain constant region.
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody.
  • the antibody fragment retains at least some of the binding activity of the parent antibody.
  • the antigen-binding fragment retains at least 10% of the parent's binding activity.
  • the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab') 2 , Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
  • Fab fragments consist of one light chain, one heavy chain CH1 and variable regions.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • Fab'fragment contains a light chain and a heavy chain part (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed between them to form F(ab') 2 molecules.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
  • domain antibody fragment is an immunoglobulin fragment with immunological function that contains only a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
  • binding to GPC3 refers to the ability to interact with human GPC3 or its epitope.
  • antigen-binding site in the present application refers to a linear or three-dimensional site recognized by the antibody or antigen-binding fragment of the present application.
  • epitope refers to a site on an antigen that binds to an antibody (or antigen-binding fragment).
  • Epitopes can be formed by adjacent amino acids, or amino acids that are close but not adjacent through the tertiary folding of the peptide chain. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope binds to a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • the term "specific binding” as used in this application refers to the binding of an antibody (or antigen-binding fragment) to an epitope on a predetermined antigen.
  • an antibody or antigen-binding fragment
  • SPR surface plasmon resonance
  • the equilibrium dissociation constant (K D ) of the antibody binds to the predetermined antigen; and the affinity of the antibody (or antigen-binding fragment) to the predetermined antigen is at least twice that of the non-specific antigen (such as BSA).
  • cross-reactivity refers to the ability of the antibody (or antigen-binding fragment) of the present application to bind to GPC3 (or its epitope) from different species. Cross-reactivity is measured by detecting specific reactivity with antigen in binding assays (such as SPR and ELISA), or binding or functional interaction with cells that physiologically express GPC3. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the inhibition/blocking of the ligand can reduce or change the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking.
  • Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-GPC3 antibody compared to ligand not contacted with anti-GPC3 antibody.
  • inhibition of growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity, which means that cells expressing Fc receptors directly kill the antibody by recognizing the Fc segment of the antibody. Coated target cells.
  • the ADCC effect function of the antibody can be enhanced or reduced or eliminated by modifying the Fc segment on the IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody.
  • mice can be immunized with human GPC3 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described in this application are genetically engineered to add one or more human FR regions to the CDR regions of non-human origin.
  • the human FR germline sequence can be obtained from the database ImmunoGeneTics (IMGT), or from The Immunoglobulin FactsBook magazine (, Academic Press, 2001ISBN012441351).
  • the engineered antibody or antigen-binding fragment of the application can be prepared and purified by conventional methods.
  • the cDNA sequence of the antibody can be cloned and recombined into an expression vector, which can stably transfect the host cell.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are expanded in the bioreactor to produce antibodies.
  • the culture solution can be purified and collected by conventional techniques. It can be filtered and concentrated by conventional methods. Common methods can also be used to remove soluble mixtures and polymers, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the monoclonal antibody (mAb) of the present application refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, such as comprising any of the antibodies of the present application, the subject has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent also referred to as "therapeutically effective amount” that is effective to alleviate the symptoms of any particular disease can vary according to various factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired effect in the subject. ability.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present application may not be effective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • Effective amount includes an amount sufficient to improve or prevent a medical disease or its symptoms.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the disease to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Exogenous refers to substances that are produced outside organisms, cells, or humans according to the background.
  • Endogenous refers to a substance produced in an organism, cell, or human body according to the background.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.
  • “Pharmaceutical composition” means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the following examples are used to further describe the application, but these examples do not limit the scope of the application.
  • the experimental methods without specific conditions in the examples of this application usually follow conventional conditions, such as Cold Spring Harbor’s antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the protein antigen is human GPC3 recombinant protein (UniProt #P51654, Gln 25 to His 559 fragment), with a His tag at the C-terminus, that is, human GPC3His protein, purchased from AcroBiosystems (Cat#GP3-H52H4).
  • CHO-K1 a monoclonal stable cell line stably expressing the full-length human or monkey (Macaca mulatta) GPC3 protein was constructed, named CHO-K1-hGPC3 and CHO-K1-cynoGPC3, respectively.
  • the cell line construction method is as follows: clone the human or monkey GPC3 full-length DNA sequence (see Table 2 below) synthesized in vitro into a lentiviral vector with puromycin resistance gene, and transfect CHO-K1 cells to obtain the cell pool
  • the F12K medium containing 8 ⁇ g/ml puromycin and 10% FBS (all three reagents were purchased from GIBCO) was used for screening culture, FACS was used to detect the expression of GPC3, and then a high-expressing monoclonal cell line was obtained by a gradient dilution method.
  • Example 2 Obtaining mouse hybridoma and antibody sequence
  • the immunogens were human GPC3-His antigen protein and/or CHO-K1-hGPC3 cells. 10 Balb/c and 5 SJL mice, and 5 C57 mice, female, 10 weeks old.
  • the immunization program and immunization program are shown in Table 3, Table 4 and Table 5 below:
  • Example 3 Use the indirect ELISA method described in Example 3 below to evaluate the serum titer of the immunized mouse serum and the ability of FACS to detect the ability to bind to cell surface antigens in Example 4(2).
  • the determination of the control titer test (greater than 100,000 times dilution) is determined Start cell fusion.
  • mice with strong serum titer, affinity and FACS binding for a final immunization kill the mice, fuse spleen cells and SP2/0 myeloma cells to obtain hybridomas; screen the target hybridomas by indirect ELISA and FACS; The strain was established as a monoclonal cell strain by the limiting dilution method.
  • the obtained positive cell lines were further screened using indirect ELISA and FACS to select hybridomas that bind to the recombinant protein.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (ThermoFisher, 15596-026) and reverse transcribed (PrimeScript TM first-strand cDNA synthesis kit, Takara #6110A).
  • the cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-primer set (Sigma #69831) and then sequenced.
  • the sequences of the mouse antibodies M1, M2, and M3 were obtained by sequencing.
  • Example 3 In vitro binding activity detection method of mouse antibody
  • Dilute GPC3His protein (AcroBiosystems, Cat#GP3-H52H4) with pH7.4 PBS to a concentration of 0.5 ⁇ g/ml, add 100 ⁇ L/well to a 96-well high-affinity ELISA plate, and incubate overnight in a refrigerator at 4°C (16 -20 hours). After washing the plate 3 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 200 ⁇ L/well of 1% bovine serum albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated at 37° C. for 0.5 hour for blocking. After blocking, discard the blocking solution, and wash the plate with PBST buffer once.
  • BSA bovine serum albumin
  • TMB chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat# S0025), incubate at 25°C for 8-15 minutes in the dark, add 50 ⁇ l/well 1M HCl to stop For the reaction, read the absorbance value at 450nm with a microplate reader (Thermo, Ascent), and analyze the data.
  • the constant region of the chimeric antibody of the present application is selected from the constant region of human IgG1 heavy chain and human kappa Chain constant region.
  • the heavy chain vector is designed as follows: signal peptide + heavy chain variable region sequence + human IgG1 constant region sequence.
  • the light chain vector is designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence.
  • the above sequences were inserted into the pCEP4 vector, and the expression vector was synthesized according to the above design. After obtaining the vector plasmid, extract the plasmid and send the plasmid to sequencing for verification.
  • the qualified plasmid was transfected into HEK293 cells (Yiqiao Shenzhou) with TF1 and cultured continuously. HEK293 cells were cultured with serum-free CD medium (Yiqiao Shenzhou, Cat#SMM 293-TI) to the logarithmic growth phase for cell transfection.
  • Chip preparation Dilute the mouse anti-human IgG (Fc) antibody with fixative reagent (10mM sodium acetate, pH 5.0) to 25 ⁇ g/mL. Each channel of the chip uses about 100 ⁇ L mouse anti-human IgG (Fc) antibody. Use 190 ⁇ L of fixation reagent to add 10 ⁇ L of mouse anti-human IgG (Fc) antibody.
  • fixative reagent 10mM sodium acetate, pH 5.0
  • Capture ligand Dilute the antibody stock solution to 4 ⁇ g/mL with running reagents, and inject it into the experimental channel (FC4) at a flow rate of 10 ⁇ L/min to capture about 200RU.
  • the reference channel (FC3) does not require ligand capture.
  • Multi-cycle analysis of analytes Dilute the human GPC3 protein by a factor of two with the running reagent, and inject the diluted sample into the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min according to the corresponding binding time and dissociation time. After each concentration analysis, the chip needs to be regenerated with 3M magnesium chloride at a flow rate of 20 ⁇ L/min for 30s to wash away the ligand and undissociated analytes. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.
  • GPC3 high-expressing cells including CHO-K1 cells (ATCC, Cat# CCL-61) overexpressing human or monkey GPC3, and human liver cancer cells JHH-7 (Nanjing Kebai, Cat# CBP60204), HepG2 (China) expressing GPC3 Cell Bank of the Academy of Sciences, Cat# SCSP-510). After trypsinization, the cells were collected by centrifugation, and the cell density was adjusted with FACS buffer (1 ⁇ PBS containing 2% FBS) and spread on a 96-well U bottom plate, 1 per well ⁇ 10 5 to 2 ⁇ 10 5 cells.
  • FACS buffer (1 ⁇ PBS containing 2% FBS
  • Centrifugation 1200g for 5 minutes, discard the supernatant, add 100 ⁇ L of antibody solution that has been gradually diluted with FACS buffer (the initial working concentration of the antibody is 100nM, 5-fold dilution, 7 concentration points, and set up 0nM points), and incubate at 4°C 1 hour.
  • Centrifugation 1200g for 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescent-labeled secondary antibody working solution prepared by FACS buffer PE anti-human IgG Fc antibody (Biolegend, Cat# 409304) or FITC anti-mouse IgG antibody (Biolegend, Cat# 406001), resuspend the cells in 100 ⁇ L per well, and incubate at 4°C for 1 hour. Centrifugation: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with PBS, they were resuspended in PBS, the fluorescence signal was detected by the flow cytometer DxFlex, and the EC 50 concentration of the antibody-bound cells was analyzed by a curve.
  • the humanization of the murine anti-human GPC3 monoclonal antibody was carried out according to the method disclosed in the literature in the field. In short, human constant domains are used to replace the parental (murine antibody) constant domains, and human antibody sequences are selected based on the homology of the murine antibody and the human antibody. In this application, the murine antibodies M1, M2, and M3 are humanized. Sourced.
  • the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain a human germline template with high homology.
  • the CDR region of the mouse antibody is transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are backmutated, and the CDR region Chemically unstable amino acid residues were optimized. After expression testing and comparison of the number of back mutations, an antibody was selected that was designed with a combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences. The sequence is as follows:
  • the humanized antibody Ab9 heavy chain variable region compared with its human germline template (germline 1) sequence, the sequence identity is 81.7%, and the sequence similarity is 87.0%.
  • the variable region of GC33 heavy chain has the same sequence similarity and identity ratio as that of Ab9.
  • Figure 1 The corresponding relationship between the humanized antibody Ab9 heavy chain variable region, GC33 antibody heavy chain variable region and its human germline template sequence is shown in Figure 1.
  • variable region of the Ab9 light chain Compared with its human germline template (germline 2), the variable region of the Ab9 light chain has a sequence identity of 88.4% and a similarity of 97.3%. Compared with its human germline template (germline 3), the GC33 light chain variable region has 86.6% sequence identity and 89.3% similarity.
  • Figure 2 for the correspondence between the Ab9 light chain variable region and its human germline template (germline 2), and the GC33 light chain variable region and its human germline template (germline 3).
  • the designed heavy chain and light chain variable region sequences are respectively connected with the heavy chain constant region and light chain constant region sequences of the human antibody.
  • the antibody heavy chain constant region is selected from: human IgG1 heavy chain constant region with ADCC enhanced after amino acid mutation, or natural human IgG1 heavy chain constant region, the sequences of which are as SEQ ID NO: 40 and SEQ ID NO, respectively : 41;
  • the light chain constant region is selected from the constant region of the human kappa chain shown in SEQ ID NO: 42.
  • the obtained heavy chain and light chain sequences are shown in Table 13, and the antibody constant region sequences are shown in Table 14.
  • the stable transfected cell line CHO-K1-human GPC3 is used for evaluation.
  • the cells were trypsinized, the cells were collected and resuspended in pre-cooled FACS buffer to adjust the cell concentration to 1 ⁇ 10 6 cells/mL.
  • FACS buffer To adjust the cell concentration to 1 ⁇ 10 6 cells/mL.
  • Take the EP tube add 1mL cell suspension, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add 1mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20 ⁇ g/ml, and incubate for 1 hour at 4°C. The supernatant was discarded by centrifugation (4°C, 1500 rpm ⁇ 5 minutes), washed twice with FACS buffer, and the supernatant was discarded.
  • Percentage of antibody endocytosis (fluorescence intensity value at each time point-average fluorescence intensity value of blank group)/(average fluorescence intensity value at zero point-average fluorescence intensity value of blank group) ⁇ 100, the results are shown in the table 18:
  • the effector cell in this experiment is NK92MI (NK92 cell overexpressing IL-2 gene, purchased from Nanjing Kebai), and the target cell is human liver cancer cell HepG2.
  • the experimental medium is RPMI1640 with 10% FBS, which is used for cell and antibody resuspension or dilution.
  • HepG2 cells were trypsinized, collected by centrifugation, resuspended in experimental medium to 1 ⁇ 10 5 cells/ml, 100 ⁇ L were plated on a white 96-well plate (Corning, 3610), incubated in a 37°C incubator for 2 days, and 50 ⁇ L 4 ⁇ Incubate the antibody at the working concentration for 15 minutes at 4°C.
  • NK92MI cells Collect NK92MI cells, resuspend them in experimental medium to 1 ⁇ 10 6 cells/ml, add 50 ⁇ L to the above reaction so that the ratio of effector cells to target cells is 5:1, and incubate in a 37°C incubator for 3-4 hours. Finally, add 100 ⁇ L Cell Titer-Glo (Promega, Cat#G7573), mix and react for 10 minutes at room temperature and avoid light, and read with a multifunctional microplate reader (Thermofisher, Lux).
  • Cell Titer-Glo Promega, Cat#G7573
  • Antibody killing percentage (%) (E-S)/(E-M) ⁇ 100
  • E is the value of the hole without antibody, that is, effector cell + tumor cell
  • S is the value of the sample well, that is, effector cell + tumor cell + antibody
  • M is the value of the medium hole.
  • the target cell of this experiment is the stable cell line CHO-K1-hGPC3 that overexpresses human GPC3.
  • the experimental culture medium is FBS-free cell culture medium F12K (GIBCO) for resuspension or dilution of cells and antibodies.
  • CHO-K1-hGPC3 cells were trypsinized, collected by centrifugation, resuspended in experimental medium to 1 ⁇ 10 5 cells/ml, spread 50 ⁇ L on a white 96-well plate (Corning, 3610), and added 25 ⁇ L 4 ⁇ working concentration of antibody (The initial working concentration of the antibody is 20nM, 5-fold dilution, 10 concentration points, and 0nM points), incubate at 37°C for 30 minutes, add 25 ⁇ L of 80% human serum (GemCell TM US, Cat#100-512), 37°C Incubate for 24 hours in an incubator. Add 50 ⁇ L CellTiter-Glo (Promega, Cat#G7573), mix well and react for 10 minutes in the dark at room temperature, and read with a multifunctional microplate reader (Thermofisher, Lux).
  • Antibody killing percentage (%) (E-S)/(E-M) ⁇ 100
  • E is the value of the well without antibody, namely cell + culture medium + human serum
  • S is the value of the sample well, namely cell + antibody + human serum
  • M is the value of medium + human serum well.
  • the panoramic isoelectric focusing (iCIEF) method was used to detect and compare the purity of the humanized antibody at the starting 0 point and the charge heterogeneity of the humanized antibody at 25°C and 40°C for one month, so as to investigate the antibody The stability.
  • the carrier amphoteric electrolyte can form a certain range of pH gradient in the tube.
  • the sample composition According to its chargeability, it migrates to the cathode or anode, and when the pH value in the capillary is the same as the isoelectric point (pI) of the component, the net charge of the solute molecule is zero, and the component will aggregate at this point in a macroscopic view without further Migration, to achieve the purpose of separating each component in a complex sample.
  • the pI value and peak ratio main peak, acid peak, alkali peak
  • System suitability sample preparation Take out the suitability sample tube from Maurice cIEF System Suitability Kit (Protein Simple, Cat#046-044), add 40 ⁇ l deionized water and 160 ⁇ l System Suitability Test Mix, mix and transfer to 1.5ml centrifugation In the tube, vortex and centrifuge, and transfer 160ul of supernatant to a 96-well sample plate for later use.
  • cIEF Master mixed solution contains 37 ⁇ l ultrapure water, 35 ⁇ l 1% MC (Protein Simple, Cat#101876), 4 ⁇ l Pharmalyte pH 3-10 (Protein Simple, Cat#17-0456-01), 2 ⁇ l 500mM arginine (Protein Simple, Cat#042-691), and the corresponding two pI Markers 6.14 (protein simple, cat:046-031) and 9.99 (protein simple, cat:046-034) 1 ⁇ l each, with a total volume of 80 ⁇ l.
  • the sample is set with 3 conditions: starting at 0 o'clock; placed in a stability test chamber (Memmer, model HPP 1060) with a humidity of 65% and a temperature of 25°C for 1 month; in the stability test chamber (Memmer, model HPP 1060) Leave it for 1 month at a humidity of 65% and a temperature of 40°C.
  • a stability test chamber Memmer, model HPP 1060
  • On-board testing open the capillary electrophoresis instrument (Protein Simple, maurice) and software, follow the instrument operation steps, perform the instrument self-test, install the capillary cartridge (Protein Simple, Cat# PS-MC02-C), and place the 96-well sample plate Enter the corresponding position of the instrument for cIEF analysis.
  • Example 11 Stability experiment of molecular variants of humanized antibodies
  • CE-SDS capillary electrophoresis
  • Sample preparation samples are taken at the initial 0 point; placed in a stability test box (Memmer, model HPP 1060) for 1 month under the conditions of a humidity of 65% and a temperature of 25°C; sampled in a stability test box (Memmer, model HPP 1060) HPP 1060) The humidity is 65% and the temperature is 40°C and placed for 1 month for sampling.
  • Non-reducing CE sample processing add samples to the EP tube, the amount of protein taken for each sample is 50 ⁇ g, add 1 ⁇ L of 10kD internal standard (Protein Simple, Cat#046-144), add 2.5 ⁇ L of 250mM IAM (Sigma, Cat #I1149-5G), add 1 ⁇ sample buffer (Protein Simple, Cat#046-567) to a final volume of 50 ⁇ L.
  • Reduce CE sample processing add samples to the EP tube, the amount of protein for each sample is 50 ⁇ g, add 1 ⁇ L of 10kD internal standard (Protein Simple, Cat#046-144), add 2.5 ⁇ L of ⁇ -mercaptoethanol (Sigma, Cat#M3148-25ML), add 1 ⁇ Sample Buffer (Protein Simple, Cat#046-567) to a final volume of 50 ⁇ L.
  • Sample test Put the 96-well sample plate into the capillary electrophoresis instrument (Protein Simple, Maurice), turn on the instrument and software, follow the instrument operation steps, perform the instrument self-test, install the capillary cartridge (Protein Simple, Cat#090-157) , Prepare the corresponding reagent and put it into the corresponding position of the instrument. According to the operation steps of the instrument, set the corresponding parameters to perform reduction or non-reduction CE analysis.
  • the non-reducing CE results showed that the main peak ratio of humanized antibody Ab9 stored at 25°C for one month was similar to that at 0 o'clock, while the main peak at 40°C for one month decreased.
  • reduced CE compared with 0 o'clock, whether it is stored at 25°C or 40°C for one month, the sum of the heavy and light chain ratios of Ab9 antibody does not change much, and the stability is good.

Abstract

提供了抗GPC3抗体CDR区的嵌合抗体、人源化抗体；还提供了包含抗GPC3抗体或其抗原结合片段的药物组合物；以及其作为抗癌药物的用途。

Description

抗GPC3抗体、其抗原结合片段及其医药用途
本申请要求2019年12月05日提交的中国专利申请(申请号201911236102.2)和2020年09月02日提交的中国专利申请(申请号202010910640.1)的优先权。
技术领域
本申请涉及一种特异性地对人GPC3具有免疫反应性的抗GPC3抗体、及其抗原结合片段、包含所述抗GPC3抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗GPC3抗体及其抗原结合片段的药物组合物，以及其作为细胞生长抑制剂和抗癌药物用途和检测或诊断肿瘤的用途。
背景技术
磷脂酰肌醇蛋白聚糖3(Glypican3，GPC3)是一种约70kd膜蛋白，属于Glypicans家族，作为器官形成中的细胞外基质蛋白而发挥细胞粘附作用、或作为细胞生长因子的受体起作用。GPC3表达之后，会被furin酶切割产生N端约40kDa可溶性部分以及约30kDa部分，通过GPI分子锚定于细胞膜的C端。
GPC3在胚胎组织(特别是肝脏和肾脏)中表达，是与器官形成相关的细胞外基质蛋白。在成人组织中，在胎盘以外观察不到GPC3的表达，但是在肝细胞癌、黑素瘤、卵巢透明细胞癌、肺鳞状细胞癌等各种癌组织中观察到表达。可见，与甲胎蛋白(α-fetoprotein；AFP)、癌胚抗原(Carcinoembryonic antigen；CEA)等蛋白质一样，GPC3也是在胚胎组织中表达的蛋白质，因此被归类为胚胎性癌抗原。即，GPC3的特征在于在正常组织细胞中不表达、但在癌细胞中特异性表达，因此可以作为癌症治疗的靶分子、肿瘤标志物。
另外，基因组学和功能研究表明，GPC3对于维持Wnt通路、Hedgehogs通路的激活有重要作用。例如，GPC3偶联的硫酸乙酰肝素分子可以增强Wnts与其受体的结合，从而对于维持Wnt通路有重要作用。GPC3表达于脑、消化道、膀胱，性腺和皮肤，并高度表达于肝细胞癌表面；肝癌发生中Wnt通路起到了重要的作用，例如20％肝细胞癌β-Catenin通路突变以及Frizzled-7受体过度表达，因此GPC3在部分肝细胞癌发生过程中可能起到了促进作用。
目前进入临床阶段并公开临床结果的的靶向GPC3大分子药物是由罗氏研发的Codrituzumab(考曲妥珠单抗；别名GC33)，专利号：US20100248359，该抗体药物靶向人GPC3的C端524-563氨基酸，属于人IgG1、kappa亚型。GC33可诱导抗体依赖的细胞毒性作用(ADCC)和补体依赖的细胞毒性作用(CDC)，在HuH-7肿瘤药效小鼠模型中，具有明显抑制肿瘤生长的效果。GC33处于临床II期(NCT01507168)，适应症有肝细胞癌(HCC)、转移性肝细胞癌、肝癌、转移性肝癌等。但由于临床药效结果不佳，罗氏停止了Codrituzumab的开发。
抗体的稳定性与其产生的药物效果、在人体内的代谢和安全性都有很大关系，例如抗体的稳定性越好，就越少产生免疫原性。
发明内容
本申请提供了一种抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区。
在一些实施方案中，本申请的抗GPC3抗体或其抗原结合片段包含选自以下的重链可变区和/或轻链可变区：
所述的重链可变区包含至少1个选自以下序列所示的HCDR1：SEQ ID NO：7，SEQ ID NO：8，
且所述的抗体重链可变区包含至少1个选自以下序列所示的HCDR2：SEQ ID NO：9，SEQ ID NO：10，SEQ ID NO：11，
且所述的抗体重链可变区包含至少1个选自以下序列所示的HCDR3：SEQ ID NO：12，SEQ ID NO：13，SEQ ID NO：14；和
所述的抗体轻链可变区包含至少1个选自以下序列所述的LCDR1：SEQ ID NO：15，SEQ ID NO：16，
且所述的抗体轻链可变区包含至少1个选自以下序列所述的LCDR2：SEQ ID NO：17，SEQ ID NO：18，
且所述的抗体轻链可变区包含至少1个选自以下序列所述的LCDR3：SEQ ID NO：19，SEQ ID NO：20。
在本申请的一些实施方案中，本申请抗体或其抗原结合片段的CDR序列可以存在1-3个氨基酸的突变，其中，1-3个氨基酸的突变是指，任意1-3个氨基酸的插入、缺失或替换；具体地，所述1-3个氨基酸的突变是能够优化抗体活性、抗体稳定性或降低免疫原性的1-3个氨基酸的插入、缺失或替换。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述的抗体重链可变区包含：
分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；
分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3。
或分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述的抗体轻链可变区包含：
分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3；
或分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：所述的重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；所述的轻链可变区包含：分别如SEQ ID NO：15SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3。
本申请还涉及一种具体方案，一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：
SEQ ID NO：7或与SEQ ID NO：7相比具有1-3个氨基酸突变的HCDR1、
SEQ ID NO：9或与SEQ ID NO：9相比具有1-3个氨基酸突变的HCDR2和
SEQ ID NO：12或与SEQ ID NO：12相比具有1-3个氨基酸突变的HCDR3；
所述的轻链可变区包含：
SEQ ID NO：15或与SEQ ID NO：15相比具有1-3个氨基酸突变的LCDR1、
SEQ ID NO：17或与SEQ ID NO：17相比具有1-3个氨基酸突变的LCDR2和
SEQ ID NO：19或与SEQ ID NO：19相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；
所述的轻链可变区包含：分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：
SEQ ID NO：7或与SEQ ID NO：7相比具有1-3个氨基酸突变的HCDR1、
SEQ ID NO：10或与SEQ ID NO：10相比具有1-3个氨基酸突变的HCDR2和
SEQ ID NO：13或与SEQ ID NO：13相比具有1-3个氨基酸突变的HCDR3；
所述的抗体轻链可变区包含：
SEQ ID NO：15或与SEQ ID NO：15相比具有1-3个氨基酸突变的LCDR1、
SEQ ID NO：17或与SEQ ID NO：17相比具有1-3个氨基酸突变的LCDR2和
SEQ ID NO：19或与SEQ ID NO：19相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：
分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3；
所述的轻链可变区包含：分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：SEQ ID NO：8或与SEQ ID NO：8相比具有1-3个氨基酸突变的HCDR1、SEQ ID NO：11或与SEQ ID NO：11相比具有1-3个氨基酸突变的HCDR2和SEQ ID NO：14或与SEQ ID NO：14相比具有1-3个氨基酸突变的HCDR3；
所述的轻链可变区包含：SEQ ID NO：16或与SEQ ID NO：16相比具有1-3个氨基酸突变的LCDR1、SEQ ID NO：18或与SEQ ID NO：18相比具有1-3个氨基酸突变的LCDR2和SEQ ID NO：20或与SEQ ID NO：20相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；所述的轻链可变区包含：分别如SEQ ID NO：16SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：SEQ ID NO：7或与SEQ ID NO：7相比具有1-3个氨基酸突变的HCDR1、SEQ ID NO：9或与SEQ ID NO：9相比具有1-3个氨基酸突变的HCDR2和SEQ ID NO：12或与SEQ ID NO：12相比具有1-3个氨基酸突变的HCDR3；所述的轻链可变区包含：SEQ ID NO：16或与SEQ ID NO：16相比具有1-3个氨基酸突变的LCDR1、SEQ ID NO：18或与SEQ ID NO：18相比具有1-3个氨基酸突变的LCDR2和SEQ ID NO：20或与SEQ ID NO：20相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；所述的轻链可变区包含：分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：SEQ ID NO：7或与SEQ ID NO：7相比具有1-3个氨基酸突变的HCDR1、SEQ ID NO：10或与SEQ ID NO：10相比具有1-3个氨基酸突变的HCDR2和SEQ ID NO：13或与SEQ ID NO：13相比具有1-3个氨基酸突变的HCDR3；所述的轻链可变区包含：SEQ ID NO：16或与SEQ ID NO：16相比具有1-3个氨基酸突变的LCDR1、SEQ ID NO：18或与SEQ ID NO：18相比具有1-3个氨基酸突变的LCDR2和SEQ ID NO：20或与SEQ ID NO：20相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3；所述的轻链可变区包含：分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
所述的重链可变区包含：SEQ ID NO：8或与SEQ ID NO：8相比具有1-3个氨基酸突变的HCDR1、SEQ ID NO：11或与SEQ ID NO：11相比具有1-3个氨基酸突变的HCDR2和SEQ ID NO：14或与SEQ ID NO：14相比具有1-3个氨基酸突变的HCDR3；所述的轻链可变区包含：SEQ ID NO：15或与SEQ ID NO：15相比具有1-3个氨基酸突变的LCDR1、SEQ ID NO：17或与SEQ ID NO：17相比具有1-3个氨基酸突变的LCDR2和SEQ ID NO：19或与SEQ ID NO：19相比具有1-3个氨基酸突变的LCDR3。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述的抗体为鼠源抗体或其片段，嵌合抗体或其片段，人抗体或其片段以及人源化抗体或其片段。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体。
在具体的实施方案中，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2或IgG4的重链恒定区。
在具体的实施方案中，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1的重链恒定区。
在具体的实施方案中，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO：41所示的重链恒定区。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结 合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含突变的IgG1重链恒定区，其相较于亲本氨基酸具有增强的ADCC毒性。在具体的实施方案中，所述突变的IgG1重链恒定区如SEQ ID NO：40所示。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链、λ链的轻链恒定区或其突变体；
优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链的轻链恒定区；
更优选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO:42所示的轻链恒定区。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自以下序列所示的重链可变区：SEQ ID NO：21，SEQ ID NO：23，SEQ ID NO：24或SEQ ID NO：26。
本申请还涉及一种优选方案，一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO：21，SEQ ID NO：23，SEQ ID NO：24或SEQ ID NO：26。
在本申请上下文中，提及“至少70％的同一性”是指，例如70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％的同一性，或任意两个数值之间的范围。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自以下序列所示的轻链可变区：SEQ ID NO：22或SEQ ID NO：25。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段的轻链可变区包含与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO：22或SEQ ID NO：25。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含：
SEQ ID NO：21所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：22所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：21所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：25所示的轻链可变区、 或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：23所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：22所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：23所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：25所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：24所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：22所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：24所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：25所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：26所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：22所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
SEQ ID NO：26所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：25所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含：
重链可变区为SEQ ID NO：21和轻链可变区为SEQ ID NO：22；
重链可变区为SEQ ID NO：21和轻链可变区为SEQ ID NO：25；
重链可变区为SEQ ID NO：23和轻链可变区为SEQ ID NO：22；
重链可变区为SEQ ID NO：23和轻链可变区为SEQ ID NO：25；
重链可变区为SEQ ID NO：24和轻链可变区为SEQ ID NO：22；
重链可变区为SEQ ID NO：24和轻链可变区为SEQ ID NO：25；
重链可变区为SEQ ID NO：26和轻链可变区为SEQ ID NO：22；或
重链可变区为SEQ ID NO：26和轻链可变区为SEQ ID NO：25。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含与以下序列相比具有至少80％，85％，90％，95％、99％或100％同一性的重链：SEQ ID NO：27，SEQ ID NO：29，SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO:36，SEQ ID NO：37，SEQ ID NO：38或SEQ ID NO：39。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含与以下序列相比具有至少80％，85％，90％，95％、99％或100％同一性的轻链：SEQ ID NO：28或SEQ ID NO：31。
在本申请的另一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含：
SEQ ID NO：27所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：27所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：29所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：29所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：30所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：30所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：32所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：32所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结 合片段，其中所述抗GPC3抗体包含：
重链为SEQ ID NO：27和轻链为SEQ ID NO：28；
重链为SEQ ID NO：27和轻链为SEQ ID NO：31；
重链为SEQ ID NO：29和轻链为SEQ ID NO：28；
重链为SEQ ID NO：29和轻链为SEQ ID NO：31；
重链为SEQ ID NO：30和轻链为SEQ ID NO：28；
重链为SEQ ID NO：30和轻链为SEQ ID NO：31；
重链为SEQ ID NO：32和轻链为SEQ ID NO：28；或
重链为SEQ ID NO：32和轻链为SEQ ID NO：31。
在本申请的一些实施方案中，提供一种如上所述的抗GPC3抗体或其抗原结合片段，其包含：
SEQ ID NO：36所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：36所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：37所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：37所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO:38所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：38所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：39所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：28所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
SEQ ID NO：39所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：31所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链。
本申请还涉及一些实施方案，提供一种如上所述的抗GPC3抗体或其抗原结 合片段，其中所述抗GPC3抗体包含：
重链为SEQ ID NO：36和轻链为SEQ ID NO：28；
重链为SEQ ID NO：36和轻链为SEQ ID NO：31；
重链为SEQ ID NO：37和轻链为SEQ ID NO：28；
重链为SEQ ID NO：37和轻链为SEQ ID NO：31；
重链为SEQ ID NO：38和轻链为SEQ ID NO：28；
重链为SEQ ID NO：38和轻链为SEQ ID NO：31；
重链为SEQ ID NO：39和轻链为SEQ ID NO：28；或
重链为SEQ ID NO：39和轻链为SEQ ID NO：31。
本申请进一步提供一种多核苷酸，其编码如上所述的抗GPC3抗体或其抗原结合片段。
本申请进一步提供一种表达载体，其含有如上所述的多核苷酸。
本申请进一步提供一种宿主细胞，其导入或含有如上所述所述的表达载体。
在本申请一个具体的实施方案中，一种如上所述的宿主细胞，其中所述的宿主细胞为细菌，具体为大肠杆菌。
在本申请一个具体的实施方案中，一种如上所述的宿主细胞，其中所述的宿主细胞为酵母菌，具体为毕赤酵母。
在本申请一个具体的实施方案中，一种如上所述的宿主细胞，其中所述的宿主细胞为哺乳动物细胞，具体为CHO细胞或HEK293细胞。
本申请进一步提供一种生产抗GPC3抗体或其抗原结合片段的方法，包括：培养上述的宿主细胞(例如HEK293细胞)；从培养物中分离抗体(例如从细胞培养液中分离抗体)；以及对所述抗体进行纯化(例如以层析方法纯化抗体)。
本申请进一步提供一种药物组合物，其含有上述所述的抗GPC3抗体或其抗原结合片段、以及可药用的赋形剂、稀释剂或载体。
在本申请的进一个具体的实施方案中，提供了一种检测或诊断试剂盒，其含有本申请所述的抗GPC3抗体或其抗原结合片段，和一种或多种能检测该抗GPC3抗体或其抗原结合片段与GPC3(或其表位)结合的试剂。
在本申请的一个具体的实施方案中，提供了一种检测或诊断试剂盒，其含有本申请所述的抗GPC3抗体或其抗原结合片段或其药学上可接受的盐、以及可用于检测或诊断的标记的第二抗体、缓冲液和底物。
在本申请一个具体的实施方案中，提供了一种如上所述的抗GPC3抗体或其抗原结合片段或如前所述的组合物在制备药物中的用途，所述药物用于治疗或预防GPC3介导的疾病或病症。
在本申请一个具体的实施方案中，一种如上所述的抗GPC3抗体或其抗原结合片段或如前所述的组合物在制备用于治疗或预防GPC3介导的疾病或病症的药物中的用途。
在本申请一个具体的实施方案中，一种如上所述的抗GPC3抗体或其抗原结合片段或如前所述的组合物在制备试剂或试剂盒中的用途，其中所述试剂或试剂盒可用于检测、诊断、预后GPC3介导的疾病或病症。
在本申请一个具体的实施方案中，一种如上所述的抗GPC3抗体或其抗原结合片段或如前所述的组合物，其用于检测、诊断、预后GPC3介导的疾病。
本申请还进一步提供一种治疗或预防GPC3介导的疾病的方法，包括步骤：向受试者提供治疗有效量或预防有效量的如上所述的抗GPC3抗体或其抗原结合片段；或者向受试者提供治疗有效量或预防有效量的如前所述的药物组合物。
本申请所述的疾病或病症为癌症；具体地，所述的疾病或病症为GPC3介导的癌症；进一步的，所述的癌症选自：乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤或黑色素瘤。
本申请提供了一种抗GPC3抗体或抗原结合片段，其能够特异性的与GPC3抗原(或其表位)、表达GPC3的细胞结合，且具有显著的CDC活性，其对肿瘤的杀伤效果显著。
另外，本申请的抗GPC3抗体或其抗原结合片段具有良好的内吞作用，适于和药物偶联。本申请的抗GPC3抗体或抗原结合片段在高抗体活性基础上，免疫原性更低，稳定性更高，并显著优于Codrituzumab。本申请抗GPC3抗体或抗原结合片段具有更好的作为抗癌药物的潜力，保证用药安全。
附图说明
图1：人源化抗体Ab9重链可变区、GC33抗体重链可变区与其人种系模板序列对应关系(注：灰色底纹标出的为抗体与相应人种系模板不一致的氨基酸)。
图2：人源化抗体Ab9轻链可变区、GC33抗体轻链可变区与其人种系模板序列对应关系(注：灰色底纹标出的为抗体与相应人种系模板不一致的氨基酸)。
具体实施方式
术语
为了更容易理解本申请，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本申请所属领域的一般技术人员通常理解的含义。
本申请所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
本申请所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。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。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本申请中，本申请所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区源自人源或鼠源的κ、λ链或其变体。
在本申请中，本申请所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区源自人源或鼠源的IgG1、IgG2、IgG 3、IgG 4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的框架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2，和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本申请所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat或Chothia或ABM定义规则。
本申请中，除非另有说明，可变区的氨基酸编号采用Kabat编号系统。本领域技术人员可以根据常识确定某特定氨基酸位置在不同编号系统中的对应关系。
术语“GPC3”包括由细胞天然表达的GPC3的任何变体或同种型。本申请的抗体(或其抗原结合片段)可与非人物种的GPC3交叉反应。作为另一种选择，该抗体也可以是人GPC3特异性的，可不表现出与其他物种的交叉反应性。GPC3或其任何变体或同种型可从细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。具体地，抗GPC3抗体靶向具有正常糖基化模式的人源GPC3(或其表位)。
术语“鼠源抗体”在本申请中为根据本领域知识和技能制备的针对人GPC3(或其表位)的单克隆抗体。制备时用GPC3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本申请一个具体的实施方案中，所述的鼠源GPC3抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本申请的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(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所引入的突变)。然而，术语“人抗体”不包括“人源化抗体”。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。人 源化抗体有助于克服嵌合抗体由于携带非人蛋白成分，从而诱导的强烈的免疫应答反应的缺点。为避免免疫原性的下降而引起活性的下降，可对所述的人源化抗体的可变区进行最少回复突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻第一物种抗体诱发的免疫应答反应。作为非限制性示例，为建立嵌合抗体，现建立分泌第一物种单抗的杂交瘤，然后从杂交瘤细胞中克隆可变区基因，再根据需要克隆第二物种抗体的恒定区基因，将可变区基因与恒定区基因连接成嵌合基因后插入载体中，最后在工业系统中表达嵌合抗体分子。恒定区可选自第二物种的IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体；具体地，包含第二物种的IgG1、IgG2或IgG4重链恒定区，或者具有增强的ADCC毒性的IgG1重链恒定区。
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合活性。通常，当基于摩尔来表示活性时，抗原结合片段保留至少10％的母体结合活性。具体地，抗原结合片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段的示例包括但不限于：Fab、Fab’、F(ab’) 2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson，2005，Nat.Biotechnol.23：1126-1136中。
“Fab片段”由一条轻链、一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fab’片段”含有一条轻链和一条重链的部分(包含VH结构域、CH1结构域、以及CH1和CH2结构域之间区域)；由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’) 2分子。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区VH和轻链可变区VL的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。
术语“单链抗体”是由抗体的重链可变区VH和轻链可变区VL通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功 能的免疫球蛋白片段。作为非限制性示例，两个或多个VH区与肽接头共价连接以形成二价结构域抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。
本申请的术语“与GPC3结合”，指能与人GPC3或其表位相互作用。
本申请的术语“抗原结合位点”指本申请抗体或抗原结合片段识别的线性或三维空间位点。
术语“表位”是指抗原上与抗体(或抗原结合片段)结合的位点。表位可以由相邻的氨基酸、或通过肽链的三级折叠而靠近但不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位与给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
本申请所用的术语“特异性结合”是指抗体(或抗原结合片段)与预定的抗原上的表位结合。通常，当使用人GPC3作为分析物并使用抗体作为配体时，在仪器中通过表面等离子体共振(SPR)技术测定，抗体(或抗原结合片段)以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合；并且，抗体(或抗原结合片段)与预定抗原结合的亲和力是其与非特异性抗原(如BSA)结合的亲和力的至少两倍。
术语“交叉反应”是指本申请的抗体(或抗原结合片段)与来自不同物种的GPC3(或其表位)结合的能力。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与抗原的特异性反应性，或与生理表达GPC3的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断两者。优选地，对配体的抑制/阻断能够降低或改变无抑制或无阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗GPC3抗体接触时，与未与抗GPC3抗体接触的配体相比，任何可测量的配体结合亲和力的降低。
术语“抑制生长”(例如涉及细胞)旨在包括任何可测量的细胞生长的降低。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。
本申请中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞，通过识别抗体的Fc段，直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段进行修饰，而增强或降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人GPC3或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本申请所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从数据库ImMunoGeneTics(IMGT)得到，或者从The Immunoglobulin FactsBook杂志(,Academic Press出版社，2001ISBN012441351)上获得。
本申请工程化的抗体或抗原结合片段可用常规方法制备和纯化。作为非限制性示例，抗体的cDNA序列可以克隆并重组至表达载体，所述表达载体可以稳定地转染宿主细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器中扩大培养以生产抗体。可以用常规技术纯化、收集培养液。可用常规方法进行过滤浓缩。也可以用常规方法去除可溶的混合物和多聚体，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本申请的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术、合成技术(如CDR-grafting)、或其它现有技术进行重组得到。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本申请的任一种抗体，所述受试者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本申请的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是 根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。
“有效量”包含足以改善或预防医学疾病或其症状的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的疾病、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要据背景在生物、细胞或人体外产生的物质。
“内源性”指根据背景在生物、细胞或人体内产生的物质。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑传代数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或其抗原结合片段，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
以下结合实施例用于进一步描述本申请，但这些实施例并非限制着本申请的范围。本申请实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1：蛋白抗原和细胞株准备
(1)蛋白抗原准备
蛋白抗原是人GPC3重组蛋白(UniProt #P51654,Gln 25至His 559片段)，C端带His标签，即人GPC3His蛋白，购买自AcroBiosystems公司(Cat # GP3-H52H4)。
表1.人GPC3(Gln 25-His 559)氨基酸序列
Figure PCTCN2020133588-appb-000001
(2)细胞株构建
以CHO-K1为宿主细胞，构建稳定表达人或猴(Macaca mulatta)GPC3全长蛋白的单克隆稳定细胞株，分别命名为CHO-K1-hGPC3和CHO-K1-cynoGPC3。细胞株构建方法如下：将体外合成的人或猴GPC3全长DNA序列(见下表2)克隆到带有嘌呤霉素抗性基因的慢病毒载体，转染CHO-K1细胞，得到的细胞池用含8μg/ml嘌呤霉素、10％FBS的F12K培养基(三种试剂均购买自GIBCO)进行筛选培养，FACS检测GPC3的表达量，然后梯度稀释法获得高表达单克隆细胞株。
表2.人GPC3和猴GPC3的DNA序列
Figure PCTCN2020133588-appb-000002
Figure PCTCN2020133588-appb-000003
Figure PCTCN2020133588-appb-000004
实施例2：鼠杂交瘤及抗体序列的获得
免疫原分别为人GPC3-His抗原蛋白和/或CHO-K1-hGPC3细胞。10只Balb/c和5只SJL小鼠，以及5只C57小鼠，雌性，10周龄。
两种免疫佐剂：
(1)使用Sigma完全弗氏佐剂(CFA)和Sigma不完全弗氏佐剂(IFA)，免疫原和免疫佐剂以1:1的比例充分混合乳化，制成稳定“油包水”液体。
(2)使用Invivo Gen的铝盐佐剂(
Figure PCTCN2020133588-appb-000005
adjuvant 2％)，按照抗原和佐剂1:1混合，形成水溶性溶液后免疫。
免疫方案和免疫程序见下表3、表4和表5：
表3.免疫方案
Figure PCTCN2020133588-appb-000006
表4.免疫程序(蛋白免疫)
Figure PCTCN2020133588-appb-000007
Figure PCTCN2020133588-appb-000008
表5.免疫程序(蛋白和细胞组合免疫)
Figure PCTCN2020133588-appb-000009
对免疫小鼠血清使用如下实施例3所述的间接ELISA法评估血清效价及实施例4(2)FACS检测结合细胞表面抗原的能力，对照效价检测情况(大于10万倍稀释度)决定启动细胞融合。
选择血清效价、亲和力及FACS结合强的免疫小鼠进行一次终免疫；处死小鼠，取脾细胞和SP2/0骨髓瘤细胞融合后获得杂交瘤；通过间接ELISA和FACS筛选到目标杂交瘤；通过有限稀释法建株为单克隆细胞株。得到的阳性细胞株进一步使用间接ELISA和FACS进行筛选，从而选定结合重组蛋白的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(ThermoFisher，15596-026)提取RNA并反转录(PrimeScript TM第一链cDNA合成试剂盒，Takara #6110A)。将反转录得到的cDNA采用小鼠Ig-引物组(Sigma #69831)进行PCR扩增后测序，通过测序获得鼠源抗体M1、M2、M3的序列。
表6.鼠单抗的重链和轻链可变区序列
Figure PCTCN2020133588-appb-000010
Figure PCTCN2020133588-appb-000011
通过对M1，M2和M3的CDR区的分析，发现M1，M2和M3的LCDR1均含有“NGN”序列片段，推测会对抗体的稳定性产生影响。在后续的嵌合抗体和人源化抗体中，将LCDR1的“NGN”序列突变为“NRN”，因此后续研究中的CDR区序列如下表所示。
表7.重链和轻链可变区的CDR序列
Figure PCTCN2020133588-appb-000012
实施例3：鼠抗的体外结合活性检测方法
体外间接ELISA结合实验：
用pH7.4的PBS将GPC3His蛋白(AcroBiosystems，Cat # GP3-H52H4)稀释至0.5μg/ml浓度，以100μL/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4PBS含0.05％Tween-20)洗板3次后，加入用PBST稀释的1％牛血清白蛋白(BSA)封闭液200μL/孔，37℃孵育0.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板1次。
用含1％BSA的PBST稀释待测抗体，100nM起始，5倍梯度稀释，11个剂量，以100μL/孔加到酶标板中，放于37℃孵育1小时。孵育结束后用PBST洗板3次， 加入200μL/孔用含1％BSA的PBST稀释的HRP标记羊抗鼠二抗(Jackson ImmunoResearch Laboratories，cat#115-035-071)或HRP标记羊抗人二抗(Rockland，cat#609-103-123)，37℃孵育0.5小时。用PBST洗板5次后，加入100μl/孔TMB显色底物(苏州亚科化学试剂股份有限公司，cat# S0025)，于25℃避光孵育8-15分钟，加入50μl/孔1M HCl终止反应，用酶标仪(Thermo,Ascent)在450nm处读取吸收值，分析数据。
做浓度-信号值曲线分析结果，如下表所示。结果显示：鼠抗体对人GPC3抗原的亲和力良好。
表8.鼠抗体对人GPC3抗原的亲和力(EC 50值)
鼠抗体 ELISA，EC 50(nM)
M1 0.05
M2 0.03
M3 0.07
实施例4：小鼠抗体嵌合化实验
将筛选得到的阳性鼠源抗体进行嵌合化，鼠源抗体可变区克隆在人抗体的恒定区得到嵌合抗体，本申请嵌合抗体的恒定区选自人IgG1重链恒定区和人kappa链恒定区。
重链载体设计如下：信号肽+重链可变区序列+人的IgG1恒定区序列。
轻链载体设计如下：信号肽+轻链可变区序列+人的Kappa恒定区序列。
分别将上述序列插入pCEP4载体，按照上述设计合成表达载体。得到载体质粒后，提取质粒，将质粒送测序验证。将验证合格的质粒用TF1转染至HEK293细胞(义翘神州)中，连续培养。将HEK293细胞用无血清CD培养液(义翘神州，Cat#SMM 293-TI)培养至对数生长期用于细胞转染。将21.4μg轻链质粒和23.6μg重链质粒溶解在10mL Reduced Serum Medium(GIBCO，31985-070)中混匀，然后加入200ug TF1，混匀，RT孵育15min，加入50mL细胞中。细胞培养条件：5％CO2，37℃，125rpm/min。培养期间，第1、3、5天加补料(义翘神州，Cat# M293-SUPI-100)，直到细胞活率低于70％，收取细胞上清，离心过滤。将离心过滤后的细胞培养液上样到Protein-A亲和层析柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、2M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，最终获得纯化的各嵌合抗体。
(1)体外蛋白结合实验：
根据实施例3的体外间接ELISA结合实验和如下所示的Biacore方法，测试嵌合抗体与人GPC3抗原的亲和力：
芯片制备：将鼠抗人IgG(Fc)抗体用固定试剂(10mM醋酸钠，pH 5.0)稀释到25μg/mL，芯片每个通道约使用100μL鼠抗人IgG(Fc)抗体，固定两个通道约使用190μL固定试剂加入10μL鼠抗人IgG(Fc)抗体。首先，CM5芯片 的表面用400mM EDC和100mM NHS以10μL/min的流速进行420s的活化。其次，将25μg/mL的鼠抗人IgG(Fc)抗体以10μL/min的流速注入到实验通道(FC4)约420s，固定量约为9000至14000RU。最后，芯片用1M乙醇胺以10μL/min进行420s封闭。参比通道(FC3)与试验通道(FC4)进行相同的操作。
捕获配体：将抗体原液用运行试剂分别稀释至4μg/mL，并以10μL/min的流速注入到实验通道(FC4)捕获约200RU。参比通道(FC3)不需要进行配体的捕获。
分析物多循环分析：将人GPC3蛋白用运行试剂进行2倍倍比稀释，将稀释后的样品依次以30μL/min的流速按照相应结合时间和解离时间注入到实验通道与参比通道。每一个浓度分析后，芯片需要用3M氯化镁以20μL/min的流速再生30s，洗掉配体以及未解离的分析物。进行下一个浓度分析时，实验通道需要重新捕获相同量的配体。
数据分析：使用Biacore T200分析软件计算每个抗体的KD值，参比通道(FC3)用于背景的扣减。
体外间接ELISA结合实验和Biacore方法测定结果如表9所示：
表9.嵌合抗体对人GPC3抗原的亲和力。
Figure PCTCN2020133588-appb-000013
ELISA和Biacore结果显示嵌合抗体与人GPC3抗原具有良好的亲和力。
(2)体外细胞结合实验：
GPC3高表达细胞，包括过表达人或猴GPC3的CHO-K1细胞(ATCC，Cat# CCL-61)，和表达GPC3的人肝癌细胞JHH-7(南京科佰，Cat# CBP60204)，HepG2(中国科学院细胞库，Cat# SCSP-510)，经胰酶消化后，离心收集细胞，用FACS缓冲液(含2％FBS的1×PBS)调节细胞密度后分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。离心：1200g、5分钟，弃上清，加入100μL已用FACS缓冲液梯度稀释的抗体溶液(抗体起始工作浓度为100nM，5倍稀释，7个浓度点，并设立0nM点)，4℃孵育1小时。离心：1200g、5分钟，弃上清，PBS洗细胞2次后，添加FACS缓冲液配制的荧光标记二抗工作液PE抗人IgG Fc抗体(Biolegend,Cat# 409304)或FITC抗小鼠IgG抗体(Biolegend,Cat# 406001)，100μL每孔重悬细胞，4℃孵育1小时。离心：1200g、5分钟，弃上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞计数仪DxFlex检测荧光信号，并作曲线分析抗体结合细胞的EC 50浓度。
表10.嵌合抗体对表达GPC3细胞的亲和力(EC 50值)
Figure PCTCN2020133588-appb-000014
结果显示：嵌合抗体与表达GPC3的细胞均具有很高的亲和力。
实施例5：小鼠抗体人源化实验
鼠源抗人GPC3单克隆抗体人源化如本领域文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列，本申请将鼠源抗体M1、M2和M3进行人源化。
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。
将鼠源抗体的CDR区移植到选择好的相应人源化模板上。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基、以及对VL和VH的构象有重要影响的残基进行回复突变、并对CDR区化学不稳定氨基酸残基优化，经表达测试和回复突变数量对比，选择出设计了人源化重链可变区HCVR和轻链可变区LCVR序列组合而成的抗体，序列如下：
表11.人源化抗体的CDR区
Figure PCTCN2020133588-appb-000015
Figure PCTCN2020133588-appb-000016
表12.人源化抗体的重链和轻链可变区序列
Figure PCTCN2020133588-appb-000017
其中，人源化抗体Ab9重链可变区与其人种系模板(种系1)序列相比，序列一致性为81.7％，序列相似性为87.0％。GC33重链可变区与其人种系模板(种系1)序列相比，序列相似性和一致性比例与Ab9相同。人源化抗体Ab9重链可变区、GC33抗体重链可变区与其人种系模板序列对应关系参见图1。
Ab9轻链可变区与其人种系模板(种系2)相比，序列一致性为88.4％，相似性为97.3％。GC33轻链可变区与其人种系模板(种系3)相比，序列一致性为86.6％，相似性为89.3％。Ab9轻链可变区与其人种系模板(种系2)、GC33轻链可变区与其人种系模板(种系3)对应关系参见图2。
将设计的重链和轻链可变区序列分别与人抗体的重链恒定区和轻链恒定区序列连接。示例性地，抗体重链恒定区选自：经氨基酸突变后具有ADCC增强的人IgG1重链恒定区、或天然的人IgG1重链恒定区，其序列分别如SEQ ID NO：40、SEQ ID NO：41所示；轻链恒定区选自序列如SEQ ID NO：42所示的人κ链的恒定区。得到的重链和轻链序列如表13所示，抗体恒定区序列如表14所示。
表13 人源化抗体的重链和轻链序列
Figure PCTCN2020133588-appb-000018
Figure PCTCN2020133588-appb-000019
Figure PCTCN2020133588-appb-000020
Figure PCTCN2020133588-appb-000021
Figure PCTCN2020133588-appb-000022
Figure PCTCN2020133588-appb-000023
表14.恒定区序列编号
Figure PCTCN2020133588-appb-000024
表15.抗体及其重链、轻链、可变区的序列编号
人源化抗体编号 HCVR LCVR HC LC
Ab1 SEQ ID NO：21 SEQ ID NO：22 SEQ ID NO：27 SEQ ID NO：28
Ab2 SEQ ID NO：23 SEQ ID NO：22 SEQ ID NO：29 SEQ ID NO：28
Ab3 SEQ ID NO：24 SEQ ID NO：25 SEQ ID NO：30 SEQ ID NO：31
Ab4 SEQ ID NO：21 SEQ ID NO：25 SEQ ID NO：27 SEQ ID NO：31
Ab5 SEQ ID NO：23 SEQ ID NO：25 SEQ ID NO：29 SEQ ID NO：31
Ab6 SEQ ID NO：24 SEQ ID NO：22 SEQ ID NO：30 SEQ ID NO：28
Ab7 SEQ ID NO：26 SEQ ID NO：22 SEQ ID NO：32 SEQ ID NO：28
Ab8 SEQ ID NO：21 SEQ ID NO：22 SEQ ID NO：36 SEQ ID NO：28
Ab9 SEQ ID NO：23 SEQ ID NO：22 SEQ ID NO：37 SEQ ID NO：28
Ab10 SEQ ID NO：24 SEQ ID NO：25 SEQ ID NO：38 SEQ ID NO：31
Ab11 SEQ ID NO：21 SEQ ID NO：25 SEQ ID NO：36 SEQ ID NO：31
Ab12 SEQ ID NO：23 SEQ ID NO：25 SEQ ID NO：37 SEQ ID NO：31
Ab13 SEQ ID NO：24 SEQ ID NO：22 SEQ ID NO：38 SEQ ID NO：28
Ab14 SEQ ID NO：26 SEQ ID NO：22 SEQ ID NO：39 SEQ ID NO：28
实施例6：人源化抗体的体外结合活性实验
使用实施例3所述的体外间接ELISA结合实验和实施例4中(1)所述的Biacore 方法，测定各人源化抗体对人GPC3抗原的亲和力，结果见表16：
表16.人源化抗体对人GPC3抗原的亲和力
Figure PCTCN2020133588-appb-000025
结果显示：人源化抗体与人GPC3抗原有良好的亲和力。
使用实施例4中(2)所述的体外细胞结合实验测定各人源化抗体对表达GPC3细胞的亲和力(EC 50)，结果见表17：
表17.人源化抗体对表达GPC3细胞的亲和力(EC 50值)
Figure PCTCN2020133588-appb-000026
结果显示：人源化抗体与表达GPC3的细胞有良好的亲和力。
实施例7：人源化抗体的内吞作用
检测本申请抗体结合GPC3后是否能和人GPC3共同内吞入细胞，用稳转细胞株CHO-K1-人GPC3进行评估。
细胞使用胰酶消化，收集细胞并用预冷的FACS缓冲液重悬，调整细胞浓度为1×10 6个/mL。取EP管，加入1mL细胞悬液，1500rpm离心5分钟后弃上清，加入1mL已经配制好的待测抗体重悬细胞，抗体的终浓度均为20μg/ml，4℃摇床孵育1小时，离心弃上清(4℃、1500rpm×5分钟)，FACS缓冲液洗涤两次，弃上清。每管加入100μL荧光标记二抗工作液PE抗人IgG Fc抗体(Biolegend,Cat# 409304)或FITC抗小鼠IgG抗体(Biolegend,Cat# 406001)，重悬细胞，4℃摇床孵育30分钟，离心弃上清(4℃、1500rpm×5分钟)，FACS缓冲液洗涤两次，弃上清。每管加入1mL预热的细胞培养基重悬细胞并混匀，分装为4管，每管200μL，分别为0分钟组，空白组，30分钟组和2小时组，取出0分钟组及空白组 置于冰上，其余放置于37℃培养箱，分别内吞30分钟、2小时，在相应时间点取出EP管，置于冰上预冷5分钟，所有处理组离心弃上清(4℃、1500rpm×5分钟)，用FACS缓冲液洗涤一次，弃上清。除0分钟组外，所有处理组EP管中加入250μL strip缓冲液，室温孵育8分钟，离心弃上清(4℃、1500rpm×5分钟)，FACS缓冲液洗涤两次，弃上清。所有处理组加入100μL PBS重悬细胞，用流式细胞仪DxFlex进行检测。
抗体的内吞百分比(％)＝(各个时间点荧光强度值-空白组的平均荧光强度值)/(零点时的平均荧光轻度值-空白组的平均荧光强度值)×100，结果见表18：
表18.人源化抗体在细胞中的内吞作用
Figure PCTCN2020133588-appb-000027
从表18结果显示：人源化抗体在稳转细胞株CHO-K1-人GPC3中有良好的内吞作用。
实施例8：人源化抗体的ADCC实验
本实验的效应细胞为NK92MI(过表达IL-2基因的NK92细胞，购买自南京科佰)，靶细胞为人肝癌细胞HepG2。实验培养基为含10％FBS的RPMI1640，用于细胞和抗体重悬或稀释。HepG2细胞经胰酶消化，离心收集，用实验培养基重悬成1×10 5个/ml，100μL铺于白色96孔板(Corning，3610)，37℃培养箱培养2天后，加入50μL 4×工作浓度的抗体，4℃孵育15分钟。收集NK92MI细胞，用实验培养基重悬成1×10 6个/ml，加入50μL至上述反应中，使得效应细胞和靶细胞的比例为5:1，37℃培养箱孵育3-4小时。最后添加100μL Cell Titer-Glo(Promega，Cat#G7573)，混匀室温避光反应10分钟，多功能酶标仪(Thermofisher，Lux)读数。
抗体杀伤百分比(％)＝(E-S)/(E-M)×100
E为不加抗体的孔的数值，即效应细胞+肿瘤细胞；
S为样品孔的数值，即效应细胞+肿瘤细胞+抗体；
M是培养基孔的数值。
结果显示：人源化抗体有显著的ADCC作用。
实施例9：人源化抗体的CDC实验
本实验的靶细胞为过表达人GPC3的稳定细胞株CHO-K1-hGPC3。实验培养 基为不含FBS的细胞培养基F12K(GIBCO)，用于细胞和抗体重悬或稀释。
CHO-K1-hGPC3细胞经胰酶消化，离心收集，用实验培养基重悬成1×10 5个/ml，50μL铺于白色96孔板(Corning，3610)，加入25μL 4×工作浓度的抗体(抗体起始工作浓度为20nM，5倍稀释，10个浓度点，并设立0nM点)，37℃孵育30分钟，加入25μL 80％人血清(GemCell TM US,Cat#100-512)，37℃培养箱培养24小时。添加50μL CellTiter-Glo(Promega，Cat#G7573)，混匀室温避光反应10分钟，多功能酶标仪(Thermofisher，Lux)读数。
抗体杀伤百分比(％)＝(E-S)/(E-M)×100
E为不加抗体孔的数值，即细胞+培养基+人血清；
S为样品孔的数值，即细胞+抗体+人血清；
M是培养基+人血清孔的数值。
表19.人源化抗体的CDC作用
人源化抗体 细胞 IC 50(nM)
Ab9 CHO-K1-hGPC3 0.308
结果显示：人源化抗体Ab9具有显著的CDC作用。
实施例10：人源化抗体的电荷异质稳定性实验
本实验通过全景式等电聚焦(iCIEF)的方法，检测并比较了人源化抗体在起始0点、以及分别在25℃和40℃放置一个月的电荷异质体纯度，以此考察抗体的稳定性。
将带有两性基团的样品、两性电解质、缓冲剂和辅助添加剂的混合物注入毛细管内，当在毛细管两端加上直流电压时，载体两性电解质可以在管内形成一定范围的pH梯度，样品组分依据其所带电性向阴极或阳极泳动，毛细管内pH值与该组分的等电点(pI)相同时，溶质分子的净电荷为零，宏观上该组分将聚集在该点不再进一步迁移，达到使复杂样品中各组分分离的目的。采集图谱后，根据Marker pI值与色谱峰的迁移时间的线性关系，从而得到供试品的pI值、峰比值(主峰、酸峰、碱峰)。主要步骤如下：
系统适应性样品准备：将Maurice cIEF System Suitability Kit(Protein Simple，Cat#046-044)里的适应性样品管取出，加入40μl去离子水和160μl System Suitability Test Mix，混匀后转移到1.5ml离心管中，涡旋震荡后离心，取160ul上清转移至96孔样品板待用。
配制cIEF Master混合溶液：包含37μl超纯水，35μl的1％MC(Protein Simple，Cat#101876)，4μl Pharmalyte pH 3–10(Protein Simple，Cat#17-0456-01)，2μl 500mM arginine(Protein Simple，Cat#042-691)，以及相应的两个pI Marker 6.14(protein simple,cat:046-031)和9.99(protein simple,cat:046-034)各1μl，总体积为80μl。
供试品制备：样品设置3个条件：起始0点；在稳定性试验箱(Memmer,型 号HPP 1060)湿度为65％和温度为25℃的条件下放置1个月；在稳定性试验箱(Memmer,型号HPP 1060)湿度为65％和温度为40℃的条件下放置1个月。取20μl相应样品，加至步骤2)中含80μl cIEF Master Mix solution的EP管中，涡旋振荡混匀后离心，取80ul上清转移至96孔样品板，离心待用。
上机检测：打开毛细管电泳仪(Protein Simple，maurice)以及软件，按照仪器操作步骤，进行仪器自检，安装毛细管卡盒(Protein Simple，Cat# PS-MC02-C)，将96孔样品板放入仪器的相应位置，进行cIEF分析。
表20.人源化抗体的iCIEF检测
Figure PCTCN2020133588-appb-000028
结果显示：与起始0点相比，Ab9在25℃放置一个月后的主峰比例变化不大，稳定性良好。在强制降解条件40℃放置一个月，抗体出现了预期的降解现象，体现在主峰比例降为26.3％；而对照抗体GC33在40℃放置一个月后主峰比例降为2.5％。
实施例11：人源化抗体的分子变异体稳定性实验
本实验通过毛细管电泳(CE-SDS)的方法，检测并比较了人源化抗体在起始0点、以及分别在25℃和40℃放置一个月的分子变异体的纯度。CE-SDS是基于蛋白样品在变性条件下凝胶电泳中的迁移，根据不同分子量的蛋白迁移时间的差异完成分离，从而得到非还原和还原处理后样品纯度的检测结果。
主要步骤如下：
样品准备：样品在起始0点取样；在稳定性试验箱(Memmer，型号HPP 1060)湿度为65％和温度为25℃的条件下放置1个月取样；在稳定性试验箱(Memmer,型号HPP 1060)湿度为65％和温度为40℃的条件下放置1个月取样。
非还原CE样品处理：向EP管中加入样品，每个样品的蛋白取用量为50μg，加入1μL的10kD内标(Protein Simple，Cat#046-144)，加入2.5μL的250mM IAM(Sigma，Cat# I1149-5G)，加入1×样本缓冲液(Protein Simple，Cat#046-567)至终体积为50μL。
还原CE样品处理：向EP管中加入样品，每个样品的蛋白取用量为50μg，加入1μL的10kD内标(Protein Simple，Cat#046-144)，加入2.5μL的β-巯基乙 醇(Sigma，Cat# M3148-25ML)，加入1×样本缓冲液(Protein Simple，Cat#046-567)至终体积为50μL。
振荡混匀后，在干式恒温器(杭州奥盛仪器有限公司，型号MK20001)中70℃加热孵育10min，之后取出，冰上孵育5min，冷却后12000rpm离心5min。离心后取35μL上清转移至仪器匹配的96孔中，之后于1000rpm离心5min。
样品检测：将96孔样品板放入毛细管电泳仪(Protein Simple，Maurice)中，打开仪器以及软件，按照仪器操作步骤，进行仪器自检，安装毛细管卡盒(Protein Simple，Cat#090-157)，准备相应的试剂放入仪器的相应位置。根据仪器操作步骤，设置相应的参数，进行还原或者非还原CE分析。
数据处理：样品检测完成后，设置相应的积分参数，通过仪器自带软件进行计算分析，得到样品纯度。
表21.人源化抗体的CE-SDS检测
Figure PCTCN2020133588-appb-000029
非还原CE结果显示，人源化抗体Ab9在25℃放置一个月的主峰比例和0点的相近，40℃放置一个月主峰有所下降。在还原CE中，与0点相比，无论是25℃还是40℃放置一个月，Ab9抗体的重轻链比例之和均变化不大，稳定性良好。

Claims (23)

  1. 一种抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    所述重链可变区包含至少1个选自以下序列所示的HCDR1：SEQ ID NO：7，SEQ ID NO：8；至少1个选自以下序列所示的HCDR2：SEQ ID NO：9，SEQ ID NO：10，SEQ ID NO：11；和至少1个选自以下序列所示的HCDR3：SEQ ID NO：12，SEQ ID NO：13，SEQ ID NO：14；且
    所述轻链可变区包含至少1个选自以下序列所示的LCDR1：SEQ ID NO：15，SEQ ID NO：16；至少1个选自以下序列所示的LCDR2：SEQ ID NO：17，SEQ ID NO：18；和至少1个选自以下序列所示的LCDR3：SEQ ID NO：19，SEQ ID NO：20。
  2. 根据权利要求1所述的抗GPC3抗体或其抗原结合片段，其中所述重链可变区包含：
    分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；或，
    分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；或，
    分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3。
  3. 根据权利要求1所述的抗GPC3抗体或其抗原结合片段，其中所述轻链可变区包含：
    分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3；或，
    分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1-3任一项所述的抗GPC3抗体或其抗原结合片段，其中：
    所述重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含：分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3，或，
    所述重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含：分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3，或，
    所述重链可变区包含：分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含：分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3，或，
    所述重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：12所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含：分别如SEQ ID NO：16 SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3，或，
    所述重链可变区包含：分别如SEQ ID NO：7、SEQ ID NO：10和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含：分别如SEQ ID NO：16、SEQ ID NO：18和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3，或，
    所述重链可变区包含：分别如SEQ ID NO：8、SEQ ID NO：11和SEQ ID NO：14所示的HCDR1、HCDR2和HCDR3；所述抗体轻链可变区包含：分别如SEQ ID NO：15、SEQ ID NO：17和SEQ ID NO：19所示的LCDR1、LCDR2和LCDR3。
  5. 根据权利要求1-4任一项所述的抗GPC3抗体或其抗原结合片段，其中所述的抗体为鼠源抗体或其片段，嵌合抗体或其片段，人抗体或其片段、以及人源化抗体或其片段。
  6. 根据权利要求1-5任一项所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体；
    优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1、IgG2或IgG4的重链恒定区；
    更优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人IgG1的重链恒定区；
    进一步优选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO：41所示的重链恒定区、或如SEQ ID NO：40所示的重链恒定区变体。
  7. 根据权利要求1-6任一项所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链、λ链的轻链恒定区或其变体；
    优选地，所述抗GPC3抗体或其抗原结合片段进一步包含源自人κ链的轻链恒定区；
    更优选地，所述抗GPC3抗体或其抗原结合片段进一步包含如SEQ ID NO:42所示的轻链恒定区。
  8. 根据权利要求1-7任一项所述的抗GPC3抗体或其抗原结合片段，其包含：
    选自以下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO：21，SEQ ID NO：23，SEQ ID NO：24或SEQ ID NO：26；
    和/或选自以下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO：22或SEQ ID NO：25。
  9. 根据权利要求1-8任一项所述的抗GPC3抗体或其抗原结合片段，其包含：
    SEQ ID NO：21所示的重链可变区和SEQ ID NO：22所示的轻链可变区；或，
    SEQ ID NO：21所示的重链可变区和SEQ ID NO：25所示的轻链可变区；或，
    SEQ ID NO：23所示的重链可变区和SEQ ID NO：22所示的轻链可变区；或，
    SEQ ID NO：23所示的重链可变区和SEQ ID NO：25所示的轻链可变区；或，
    SEQ ID NO：24所示的重链可变区和SEQ ID NO：22所示的轻链可变区；或，
    SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区；或，
    SEQ ID NO：26所示的重链可变区和SEQ ID NO：22所示的轻链可变区；或，
    SEQ ID NO：26所示的重链可变区和SEQ ID NO：25所示的轻链可变区。
  10. 根据权利要求8或9所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自以下序列所示的重链，或包含与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO：27，SEQ ID NO：29，SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO:36，SEQ ID NO：37，SEQ ID NO：38或SEQ ID NO：39。
  11. 根据权利要求8或9所述的抗GPC3抗体或其抗原结合片段，其中所述抗GPC3抗体或其抗原结合片段包含选自以下序列所示的轻链，或包含与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO：28或SEQ ID NO：31。
  12. 根据权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段，其包含：
    SEQ ID NO：27所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：27所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：29所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：29所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：30所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：30所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：32所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：32所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：36所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：36所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：37所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：37所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO:38所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：38所示的重链，和SEQ ID NO：31所示的轻链；或，
    SEQ ID NO：39所示的重链，和SEQ ID NO：28所示的轻链；或，
    SEQ ID NO：39所示的重链，和SEQ ID NO：31所示的轻链。
  13. 一种多核苷酸，其编码权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段。
  14. 一种表达载体，其含有权利要求13所述的多核苷酸。
  15. 一种宿主细胞，其导入或含有权利要求14所述的表达载体；
    优选地，所述宿主细胞为细菌、酵母菌或哺乳动物细胞；优选大肠杆菌、毕赤酵母、CHO细胞或HEK293细胞。
  16. 一种生产抗GPC3抗体或其抗原结合片段的方法，包括：
    培养权利要求15所述的宿主细胞，优选HEK293细胞；
    从培养物中分离抗体，优选从细胞培养液中分离抗体；以及
    对所述抗体进行纯化，优选地，以层析方法纯化所述抗体。
  17. 一种药物组合物，其含有：
    权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段、以及
    可药用的赋形剂、稀释剂或载体。
  18. 一种检测或诊断试剂盒，其含有：
    权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段，
    任选地，还包含一种或多种能检测所述抗GPC3或其抗原结合片段与GPC3或其表位结合的试剂。
  19. 选自以下的任一项在制备用于治疗或预防GPC3介导的疾病或病症的药物中的用途：
    权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段、权利要求17所 述的药物组合物。
  20. 选自以下的任一项在制备试剂盒中的用途：
    权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段、权利要求17所述的药物组合物；
    其中所述试剂盒用于检测、诊断、预后GPC3介导的疾病或病症。
  21. 根据权利要求19或20所述的用途，其中：
    所述疾病或病症为癌症；
    优选地，所述疾病或病症为表达GPC3的癌症；
    更优选地，所述癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
  22. 根据权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段或如权利要求17所述的药物组合物，其用于检测、诊断、预后GPC3介导的疾病；优选地，所述疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
  23. 一种治疗或预防GPC3介导的疾病的方法，包括如下步骤：
    向受试者提供治疗有效量或预防有效量的权利要求1-12任一项所述的抗GPC3抗体或其抗原结合片段；或者
    向受试者提供治疗有效量或预防有效量的权利要求17所述的药物组合物；
    优选地，所述GPC3介导的疾病选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
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