WO2024067764A1 - 抗gpc3单克隆抗体/双特异性抗体或其抗原结合片段及其用途 - Google Patents

抗gpc3单克隆抗体/双特异性抗体或其抗原结合片段及其用途 Download PDF

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WO2024067764A1
WO2024067764A1 PCT/CN2023/122405 CN2023122405W WO2024067764A1 WO 2024067764 A1 WO2024067764 A1 WO 2024067764A1 CN 2023122405 W CN2023122405 W CN 2023122405W WO 2024067764 A1 WO2024067764 A1 WO 2024067764A1
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amino acid
acid residues
seq
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PCT/CN2023/122405
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牟宗春
李宁
刘小红
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信立泰(成都)生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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

  • the present invention relates to a monoclonal antibody (particularly a mouse, chimeric or humanized monoclonal antibody)/bispecific antibody or an antigen-binding fragment thereof, which specifically binds to human GPC3 with high affinity and functional activity.
  • the present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment, an expression vector, a host cell and a method for expressing the antibody or antigen-binding fragment.
  • the present invention further provides a drug conjugate, a monoclonal antibody, a bispecific molecule and a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and a diagnostic and therapeutic method using the anti-GPC3 monoclonal antibody/bispecific antibody or antigen-binding fragment thereof of the present invention.
  • GPC3 is a heparan sulfate proteoglycan expressed on the surface of a variety of malignant cells, such as hepatocellular carcinoma (HCC) cells.
  • HCC hepatocellular carcinoma
  • Glypican-3 is linked to the cell surface via a glycosyl-phosphatidylinositol anchor (GPI).
  • GPC3 has been shown to be highly expressed in more than 70% of HCC biopsies but not in adjacent non-tumor tissues. The disease-free survival rate of GPC3-positive HCC patients is significantly lower than that of GPC3-negative HCC patients.
  • GPC3 has cell growth-inhibitory activity through antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity (International Patent Application WO 2003/000883).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • GPC3 is cleaved in vivo and secreted into the blood as a secreted form of GPC3, and tumor diagnosis can be performed using antibodies that can detect the secreted form of GPC3 (International Patent Applications WO 2004/022739, WO 03/100429 and WO 2004/018667).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Patent CN1842540B discloses an anti-GPC3 antibody that has higher ADCC and CDC activities than traditional antibodies, but its binding ability to the GPC3 epitope is still weak;
  • Patent CN104520331B discloses a high-affinity antibody specific for glypican 3 (GPC3). Although its binding ability to the GPC3 epitope has been improved, its cytotoxicity still needs to be improved.
  • Bispecific antibodies that simultaneously recognize dual epitopes of GPC3 can have a stronger binding ability to tumor cells, thereby enabling the development of more lethal drug compositions.
  • Antibody-drug conjugate is a new type of targeted drug therapy, which is formed by conjugating antibodies with small molecule drugs with strong cytotoxicity. It has both the powerful lethality of small molecule drugs and the high targeting of monoclonal antibodies, and has become a hot spot in the research and development of tumor targeted therapy.
  • ADC generally includes three parts connected in a certain way: antibody or antibody-like ligand, linker and small molecule drug. The targeting of ADC comes from the antibody part, and the toxicity mainly comes from small molecule drugs. The antibody part can also be toxic. After the antibody part binds to the antigen on the surface of the tumor cell, it is internalized into the cell.
  • ADC has the following characteristics: strong therapeutic efficacy; high tumor cell specificity, low false positive rate, and larger treatment safety window; weak immunogenicity, not easy to develop drug resistance; long circulation time in serum (shorter than naked antibody); weak toxicity to non-target cells.
  • the present invention provides an anti-GPC3 monoclonal/bispecific antibody or an antigen-binding fragment thereof, such as a mouse, human, chimeric or humanized monoclonal antibody or an antigen-binding fragment thereof, and a bispecific antibody containing two antigen-binding domains, wherein the antibody or the antigen-binding fragment thereof binds to the GPC3 protein, and the antibody or the antigen-binding fragment thereof has good GPC3 protein binding affinity.
  • an anti-GPC3 monoclonal/bispecific antibody or an antigen-binding fragment thereof such as a mouse, human, chimeric or humanized monoclonal antibody or an antigen-binding fragment thereof, and a bispecific antibody containing two antigen-binding domains, wherein the antibody or the antigen-binding fragment thereof binds to the GPC3 protein, and the antibody or the antigen-binding fragment thereof has good GPC3 protein binding affinity.
  • the present invention provides an anti-GPC3 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof binds to the GPC3 protein.
  • the present invention provides an anti-GPC3 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: the anti-GPC3 monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a chain variable region (VL);
  • VH heavy chain variable region
  • VL chain variable region
  • the heavy chain variable region comprises a CDR region, wherein the CDR region comprises a CDR region identical to any one of the amino acid sequences SEQ ID Nos: 14 or SEQ ID Nos: 16, wherein the CDR region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact;
  • the light chain variable region comprises a CDR region, which comprises a CDR region identical to any one of the amino acid sequences SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.
  • the CDR region of the heavy chain variable region (VH) is selected from:
  • amino acid residues 26-33, 51-58 and 97-104 amino acid residues 31-35, 50-66 and 99-104; or amino acid residues 26-32, 52-57 and 99-104; or amino acid residues 26-35, 50-59 and 99-104; or any group of amino acid residues 30-35, 47-59 and 97-103 of SEQ ID Nos: 16;
  • the CDR region of the light chain variable region (VL) is selected from:
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 15;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 17;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 44;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 46;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 56;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 57;
  • the CDR region of the heavy chain variable region (VH) is selected from:
  • the CDR region of the light chain variable region (VL) is selected from:
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 15;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 44;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 45;
  • the heavy chain variable region comprises a sequence identical to the amino acid sequence SEQ ID Nos: 14 or SEQ ID Nos: 16;
  • the light chain variable region comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60.
  • the anti-GPC3 monoclonal antibody or its antigen-binding fragment further comprises a heavy chain (H) and a light chain (L), and the heavy chain (H) comprises a sequence identical to the amino acid sequence SEQ ID Nos: 61 or SEQ ID Nos: 71;
  • the light chain (L) comprises the same sequence as SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64, SEQ ID Nos: 65, SEQ ID Nos: 66, SEQ ID Nos: 67, SEQ ID Nos: 68, SEQ ID Nos: 69 or SEQ ID Nos: 70.
  • the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof further comprises a heavy chain (H) and a light chain (L), wherein the heavy chain (H) comprises a sequence identical to the amino acid sequence SEQ ID Nos: 61;
  • the light chain (L) comprises a sequence identical to SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64 or SEQ ID Nos: 65.
  • the present invention also provides an anti-GPC3 bispecific antibody (e.g., a mouse, chimeric or humanized antibody) or an antigen-binding fragment thereof, which binds to a GPC3 protein, and which contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain.
  • an anti-GPC3 bispecific antibody e.g., a mouse, chimeric or humanized antibody
  • an antigen-binding fragment thereof which binds to a GPC3 protein, and which contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain.
  • first GPC3 antigen binding domain and the second GPC3 antigen binding domain may both comprise or consist of the following: (a) heavy chain variable region (VH) and light chain variable region (VL); (b) single chain antibody (scFv); (c) diabody; (d) small antibody; (e) F(ab')2; (f) F(ab). Other known antibody derivatives in the domain, etc.
  • the first GPC3 antigen binding domain and the second GPC3 antigen binding domain may be or consist of the following: an animal-derived antibody, a humanized antibody, a fully human antibody, a chimeric antibody, or an affinity-optimized antibody. More preferably, it is a humanized antibody, a human-animal chimeric antibody (such as a human-mouse chimeric antibody), and more preferably a fully human antibody.
  • the antibody may be any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD antibodies or antibodies of other subtypes. Preferably, it is an IgG1, IgG2 or IgG4 subtype.
  • the IgG1 antibody is an hIgG1 antibody or a variant thereof, and the amino acid sequence of the hIgG1 described in some embodiments is as shown in SEQ ID NO: 30, or has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 30;
  • first GPC3 antigen binding domain and the second GPC3 antigen binding domain specifically bind to different epitopes of GPC3;
  • connection relationship between the first GPC3 antigen binding domain and the second GPC3 antigen binding domain is as follows (a1) or (a2):
  • the linkers L1 and L2 are independently selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6;
  • the scFv is further connected to the heavy chain or light chain end of the first or second GPC3 antigen binding domain via a linker L2;
  • the heavy chain or light chain terminus is selected from the heavy chain or light chain C-terminus or N-terminus.
  • the anti-GPC3 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain
  • the structure of the first polypeptide chain can be selected from any one of the following (b1)-(b8):
  • [L1] and [L2] independently represent a linker, and the linker is selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; the CH1 is the antibody heavy chain constant region 1; the [Fcx] represents the Fc domain;
  • [AVH] and [AVL] represent the heavy chain variable region (AVH) and light chain variable region (AVL) of the first GPC3 antigen-binding domain, respectively;
  • [BVH] and [BVL] represent the heavy chain variable region (BVH) and light chain variable region (BVL) of the second GPC3 antigen-binding domain, respectively;
  • [Fcx] indicates the Fc domain, which includes CH2 and CH3;
  • the structure of the second polypeptide chain may be selected from the following (c1) or (c2):
  • [CL] is the antibody light chain constant region
  • [AVL]-[CL] represents the light chain variable region (AVL) and the light chain constant region (CL) of the first GPC3 antigen-binding domain;
  • [BVL]-[CL] represents the light chain variable region (BVL) and the light chain constant region (CL) of the second GPC3 antigen-binding domain.
  • the first polypeptide chain (heavy chain) and the second polypeptide chain (light chain) are connected via a disulfide bond.
  • the CDR region of the first heavy chain variable region is selected from the CDR region of any one of SEQ ID Nos: 14, 16, 34, 49 or 51, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the first light chain variable region is selected from the CDR region of any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the second heavy chain variable region is selected from the CDR region of any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the second light chain variable region is selected from the CDR region of any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48, and the CDR region includes CDR1 region, CDR2 region and CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.
  • the CDR region of the first heavy chain variable region is selected from:
  • amino acid residues 26-33, 51-58 and 97-104 of SEQ ID Nos: 14 (h1B12); or amino acid residues 31-35, 50-66 and 99-104 amino acid residues at positions 26-32, 52-57 and 99-104; or amino acid residues at positions 26-35, 50-59 and 99-104; or any group of amino acid residues at positions 30-35, 47-59 and 97-103;
  • the CDR region of the first light chain variable region (AVL) is selected from:
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 15(h1B12);
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 17(m1B12);
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 46 (h1B12-G34A);
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 56;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 57;
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 60;
  • CDR region of the second heavy chain variable region is selected from:
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 1(YP7);
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 3 (hYP7);
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 6(YP8);
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 26-33, 51-60 and 99-106; or amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 8(YP9);
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 26-33, 51-60 and 99-106; or amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 12 (YP9.1);
  • the CDR region of the second light chain variable region (BVL) is selected from:
  • the CDR region of the first heavy chain variable region is selected from: amino acid residues 26-33, 51-58 and 97-104; or amino acid residues 31-35, 50-66 and 99-104; or amino acid residues 26-32, 52-57 and 99-104; or amino acid residues 26-35, 50-59 and 99-104; or any group of amino acid residues 30-35, 47-59 and 97-103 of SEQ ID Nos: 14 (h1B12).
  • the CDR region of the first light chain variable region (AVL) is selected from:
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 15(h1B12);
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 44 (h1B12-G34R);
  • the CDR region of the second heavy chain variable region (BVH) is selected from:
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 3 (hYP7);
  • amino acid residues 26-33, 51-58 and 97-103 amino acid residues 31-35, 50-66 and 99-103; or amino acid residues 26-32, 52-57 and 99-103; or amino acid residues 26-35, 50-59 and 99-103; or any group of amino acid residues 30-35, 47-59 and 97-102 of SEQ ID Nos: 20(m2D8);
  • the CDR region of the second light chain variable region (BVL) is selected from:
  • amino acid residues 27-38, 56-57 and 95-103 amino acid residues 24-40, 56-62 and 95-103; or any group of amino acid residues 30-42, 52-61 and 95-102 of SEQ ID Nos: 4 (hYP7).
  • the CDR region of the first heavy chain variable region is selected from positions 26-33, 51-58 and 97-104 of SEQ ID Nos: 14 (h1B12). amino acid residues at positions 26-35, 50-59 and 99-104; or amino acid residues at positions 26-35, 50-59 and 99-104; or any group of amino acid residues at positions 30-35, 47-59 and 97-103;
  • the CDR region of the first light chain variable region is selected from: amino acid residues 27-37, 55-56 and 94-102; or amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID Nos: 44 (h1B12-G34R);
  • the CDR region of the second heavy chain variable region (BVH) is selected from:
  • amino acid residues 26-33, 51-60 and 99-106 amino acid residues 31-35, 50-68 and 101-106; or amino acid residues 26-32, 52-59 and 101-106; or amino acid residues 26-35, 50-61 and 101-106; or any group of amino acid residues 30-35, 47-61 and 99-105 of SEQ ID Nos: 5 (hYP7HM);
  • amino acid residues 26-33, 51-58 and 97-103 amino acid residues 31-35, 50-66 and 99-103; or amino acid residues 26-32, 52-57 and 99-103; or amino acid residues 26-35, 50-59 and 99-103; or any group of amino acid residues 30-35, 47-59 and 97-102 of SEQ ID Nos: 18 (h2D8);
  • amino acid residues 26-33, 51-58 and 97-103 amino acid residues 31-35, 50-66 and 99-103; or amino acid residues 26-32, 52-57 and 99-103; or amino acid residues 26-35, 50-59 and 99-103; or any group of amino acid residues 30-35, 47-59 and 97-102 of SEQ ID Nos: 20(m2D8);
  • the CDR region of the second light chain variable region (BVL) is selected from:
  • amino acid residues 27-37, 55-56 and 94-102 amino acid residues 24-39, 55-61 and 94-102; or any group of amino acid residues 30-41, 51-60 and 94-101 of SEQ ID No: 19 (h2D8);
  • the CDR region of the first heavy chain variable region is selected from: the CDR region of the heavy chain variable region of any one of h1B12 or m1B12, the CDR region comprising a CDR1 region, a CDR2 region and a CDR3 region, each CDR region being defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the first light chain variable region is selected from the CDR region of the light chain variable region of any one of h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A or m1B12, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the second heavy chain variable region is selected from: the CDR region of the heavy chain variable region of any one of YP7, hYP7, hYP7HM, YP8, YP9, YP9.1, h2D8 or m2D8, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;
  • the CDR region of the second light chain variable region is selected from: the CDR region of the light chain variable region of any one of YP7, hYP7, YP8, YP9, YP9.1, h2D8 or m2D8, and the CDR region includes CDR1 region, CDR2 region and CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.
  • the first heavy chain variable region is selected from any one of SEQ ID Nos: 14, 16, 34, 49 or 51;
  • the first light chain variable region (AVL) region is selected from any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60;
  • the second heavy chain variable region is selected from any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47;
  • the second light chain variable region is selected from any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48.
  • the first polypeptide chain of the anti-GPC3 bispecific antibody comprises: any one of SEQ ID Nos: 36; SEQ ID Nos: 37; SEQ ID Nos: 38; SEQ ID Nos: 39; SEQ ID Nos: 40; SEQ ID Nos: 41; SEQ ID Nos: 42 or SEQ ID Nos: 43.
  • the second polypeptide chain of the bispecific anti-GPC3 antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein VL comprises: any one of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60, and CL comprises: SEQ ID Nos: 55;
  • the second polypeptide chain of the bispecific anti-GPC3 antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein VL comprises: SEQ ID Nos: 15; SEQ ID Nos: 17; SEQ ID Nos: 44; any one of SEQ ID Nos: 45 or SEQ ID Nos: 46, and CL comprises: SEQ ID Nos: 55;
  • the present invention further provides a chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof described in the present invention.
  • the present invention also provides a polynucleotide, characterized in that the polynucleotide encodes the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention.
  • the polynucleotide of the present invention can be, for example, DNA or RNA, And may or may not contain intron sequences.
  • the polynucleotide is a cDNA molecule.
  • the present invention also provides an expression vector, characterized in that the expression vector comprises the polynucleotide of the present invention.
  • the expression vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • the present invention further provides a host cell, characterized in that the host cell comprises the polynucleotide of the present invention or the expression vector of the present invention.
  • the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, a NSO cell or other mammalian cell, preferably a CHO cell.
  • the present invention further provides an antibody-drug conjugate, characterized in that it comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof described in the present invention or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof described in the present invention and a drug or toxin.
  • the drug or toxin is selected from: one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.
  • the anti-GPC3 bispecific antibody can be prepared according to a method comprising the following steps:
  • step 2) transfecting or transforming the expression vector constructed in step 1) into host cells, and culturing the host cells;
  • sequence of the DNA molecule of the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention can be obtained by conventional techniques, such as hybridoma PCR amplification or phage display library screening, etc.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody (such as scFV).
  • the relevant sequence can be cloned into a vector, then transferred into a host bacterium, and then the relevant vector can be extracted from the host bacterium by conventional methods.
  • DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis.
  • mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell (such as bacteria), a lower eukaryotic cell (such as yeast), or a higher eukaryotic cell (such as mammalian cell).
  • Preferred animal cells include (but are not limited to): CHO, HEK-293 cells.
  • the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention can be expressed intracellularly, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art.
  • the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention, and then purified by conventional immunoglobulin purification steps, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis and other conventional separation and purification means and a combination of these methods to obtain the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention.
  • conventional immunoglobulin purification steps such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis and other conventional separation and purification means and a combination of these methods to obtain the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention.
  • the method for separating and purifying the anti-GPC3 bispecific antibody or antigen-binding fragment thereof is protein A affinity chromatography, cation exchange method or anion exchange method.
  • the resulting monoclonal antibodies or bispecific antibodies can be characterized by conventional means.
  • the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • the binding affinity of the antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:
  • a conjugated moiety selected from the group consisting of a drug, a toxin, a detectable marker, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.
  • the radionuclides include:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • therapeutic isotopes wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.
  • the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I
  • the conjugated moiety is a drug or a toxin.
  • the drug can be any cytotoxic, cell growth inhibiting or immunosuppressive drug.
  • a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl or keto group that can form a bond with the linker.
  • the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, DNA Alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors
  • typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids, or a combination thereof.
  • PBDs pyrrolo[1,4]benzodiazepines
  • indolinobenzodiazepines and oxazolidinobenzodiazepines vinca alkaloids
  • the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, cu
  • the drug or toxin is selected from: one or more of SN-38 (NK012, CAS No.: 86639-52-3), MMAE (Monomethyl auristatin E, CAS No.: 474645-27-7), PBD dimer (SG3199, CAS No.: 1595275-71-0), DX-8951 (Exatecan, CAS No.: 171335-80-1) or DUBA (duocarmycin-hydroxybenzamide-azaindole).
  • SN-38 CAS No.: 86639-52-3
  • MMAE Monomethyl auristatin E, CAS No.: 474645-27-7
  • PBD dimer SG3199, CAS No.: 1595275-71-0
  • DX-8951 Exatecan, CAS No.: 171335-80-1
  • DUBA duocarmycin-hydroxybenzamide-azaindole
  • the antibody can be coupled to the drug to form an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the ADC comprises an anti-GPC3 bispecific antibody, monoclonal antibody or antigen binding fragment of the present invention, connected to a drug or toxin via a linker.
  • the linker can be a degradable or non-degradable linker.
  • Degradable linkers are typically easily degraded in the intracellular environment, thereby releasing the therapeutic agent from the antibody.
  • Suitable degradable linkers include enzyme-degradable linkers, such as linkers containing peptidyl that can be degraded by intracellular lysosomal proteases, or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidase.
  • Peptide linkers can include dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include pH-sensitive linkers (such as hydrazone linkers that hydrolyze when pH is less than 5.5), and linkers that degrade under reducing conditions (such as disulfide linkers).
  • Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.
  • the linker Prior to attachment to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and attachment is achieved via the active reactive group.
  • Sulfhydryl-specific active reactive groups are preferred, such as maleimide compounds, halogenated amides, halogenated esters, halogenated methyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfides, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonates.
  • the linker can include, for example, maleimide attached to the antibody via thiosuccinimide.
  • the drug or toxin connected to the linker is selected from: CL2A-SN-38 (CAS No.: 1279680-68-0), mc-vc-PAB-MMAE (CAS No.: 646502-53-6), Tesirine (SG3249, CAS No.: 1595275-62-9), Deruxtecan (CAS No.: 1599440-13-7), Vc-seco-DUBA (SYD985, CAS No.: 1345681-58-4).
  • CL2A-SN-38 CAS No.: 1279680-68-0
  • mc-vc-PAB-MMAE CAS No.: 646502-53-6
  • Tesirine SG3249, CAS No.: 1595275-62-9
  • Deruxtecan CAS No.: 1599440-13-7
  • Vc-seco-DUBA SYD985, CAS No.: 1345681-58-4.
  • the molecular structure is shown in the figure below:
  • the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to SN-38 via a CL2A linker.
  • the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to MMAE via a mc-VC-PAB linker.
  • the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.
  • the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to DX-8951 (DXd) via a maleimide-GGFG linker.
  • the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to DUBA via a Vc-seco linker.
  • the antibody-drug conjugate is prepared according to a method comprising the following steps:
  • interchain disulfide bonds of any of the aforementioned anti-GPC3 bispecific antibodies are reduced to generate 2n (e.g., 2, 4, 6, 8) thiol groups;
  • the drug-linker compound is cross-linked with the reduced antibody thiol group to generate the corresponding antibody-drug conjugate;
  • the product was further purified by ultrafiltration and desalting.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-GPC3 monoclonal antibody or its antigen-binding fragment or the anti-GPC3 bispecific antibody or its antigen-binding fragment, or the antibody-drug conjugate, or the oncolytic virus, and a pharmaceutically acceptable carrier.
  • the composition comprises more than one antibody (or its antigen-binding fragment, or antibody-drug conjugate, or oncolytic virus)
  • the antibody or its antigen-binding fragment, or antibody-drug conjugate, or oncolytic virus
  • the composition may optionally contain one or more additional active pharmaceutical ingredients, such as another antibody or drug, such as an anti-tumor drug.
  • the pharmaceutical composition may contain any number of excipients.
  • Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
  • the present invention provides a nucleotide molecule encoding an anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid molecule may be present in intact cells or cell lysates, or in partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “considered substantially pure” when it is separated from other cellular components or other contaminants (such as other nucleic acids or proteins in the cell) using standard techniques, including alkaline/SDS lysis, cesium chloride density gradient centrifugation (CsCl banding), column chromatography, agarose gel electrophoresis, and other methods known in the art. See F. Ausubel et al.
  • the nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences.
  • the nucleic acid is a cDNA molecule.
  • the present invention provides a vector comprising the nucleotide of the present invention, wherein the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • Another aspect of the present invention provides a host cell comprising the above-mentioned vector or antibody; the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, a NSO cell or other mammalian cell, preferably a CHO cell.
  • the anti-GPC3 bispecific antibody or antigen-binding fragment thereof disclosed in the present invention can also be used to prepare a chimeric antigen receptor (CAR; also known as a chimeric T cell receptor, an artificial T cell receptor or a chimeric immune receptor).
  • CAR chimeric antigen receptor
  • Another aspect of the present invention provides use of the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof, antibody-drug conjugate, nucleotide, vector or host cell of the present invention in the preparation of a therapeutic drug or diagnostic reagent for autoimmune diseases, viral infections or cancer, wherein the cancer is liver cancer, gastric cancer, colorectal cancer, lung cancer or ovarian cancer, or any other type of cancer expressing GPC3.
  • the present invention also provides a method for treating a subject suffering from cancer, such as liver cancer, by selecting a subject suffering from cancer expressing GPC3, and administering to the subject a therapeutically effective amount of an anti-GPC3 bispecific antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody.
  • the present invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an effective dose of a drug or a composition thereof comprising the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention.
  • the tumor may be a solid tumor or a non-solid tumor, including but not limited to liver cancer, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer and nasopharyngeal carcinoma.
  • the method comprises administering a composition of the present invention, an anti-GPC3 bispecific antibody or antigen-binding fragment thereof, an antibody-drug conjugate, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule of the same substance that can be expressed in the subject.
  • At least one additional anti-tumor antibody can be administered in combination with the anti-GPC3 bispecific antibody of the present invention or its antigen-binding fragment, wherein the at least one additional anti-tumor antibody can be selected from anti-VISTA antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CD40 antibodies, anti-TIM-3 antibodies, anti-STAT3 antibodies and/or anti-ROR1 antibodies.
  • the antibody of the present invention or its antigen-binding fragment is administered in combination with a chemotherapeutic agent, which can be a cytotoxic agent such as epirubicin, oxaliplatin and/or 5-fluorouracil (5-FU).
  • the antibody of the present invention can be a mouse, human, chimeric or humanized antibody.
  • GPC3 also known as glypican 3 is a member of the heparan sulfate proteoglycans, anchored to the cell membrane surface by glycosyl-phosphatidylinositol (GPI).
  • the human GPC3 gene is located on chromosome X (Xp26) and encodes a 70 kDa protein containing 580 amino acids, which is cleaved endonucleoside by a furin-like convertase between Arg358 and Ser359 to produce a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, with two heparan sulfate (HS) chains on the C-terminal subunit.
  • HS heparan sulfate
  • an antibody that "specifically binds to human GPC3" refers to an antibody that binds to human GPC3 protein (and potentially GPC3 proteins from one or more non-human species) but does not substantially bind to non-GPC3 proteins.
  • antibody as referred to herein includes complete antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains thereof.
  • Complete antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains connected by disulfide bonds between the chains.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH region and the VL region can be further subdivided into high Variable regions (called complementarity determining regions (CDRs)), separated by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • bispecific antibody refers to an antibody molecule that can bind to two independent antigens or has binding specificity to different epitopes within the same antigen.
  • one antigen-binding fragment of the bispecific antibody molecule binds to the first antigen-binding domain of GPC3, and the other antigen-binding fragment binds to the second antigen-binding domain of GPC3, which can further enhance the affinity with the cell surface GPC3 antigen.
  • an antibody portion refers to one or more fragments of an antibody that specifically binds to an antigen (e.g., a GPC3 protein). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody.
  • binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL, and CH1 domain
  • the two domains VL and VH of the FV fragment are encoded by separate genes, they can be connected by a linker using recombinant methods to make them a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
  • single-chain Fv single-chain Fv
  • Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody.
  • These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragment screening for use is the same as that for intact antibodies.
  • scFv refers to a genetically engineered molecule comprising the variable region of the light chain and the variable region of the heavy chain of an immunoglobulin, connected by a suitable polypeptide linker as a genetically fused single chain antibody.
  • Fc region refers to the Fc fragment region of an antibody molecule, includes a portion of the hinge region, CH2 and CH3 domains, and has a molecular weight of about 50,000 Da.
  • the human IgG heavy chain Fc region is from Threonine at position 225 to the C-terminus.
  • IgG1 refers to a portion of a human or murine heavy chain constant region, wherein “hIgG1” refers to a portion of a human heavy chain constant region, and the “IgG1" or “hIgG1” includes CH1, CH2, and CH3 domains;
  • Fab a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, can be generated by digesting an intact antibody with the enzyme papain to produce an intact light chain and a portion of one heavy chain.
  • Fab' refers to a fragment of an antibody molecule that can be obtained by treating an intact antibody with pepsin followed by reduction to produce an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule.
  • (Fab')2 refers to an antibody fragment that can be obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds.
  • Fv refers to a gene fragment containing a light chain variable region and a heavy chain variable region expressed as two chains.
  • h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S or h1B12-N33R refers to an antibody or an antigen-binding fragment thereof in which the 34th position of the light chain of h1B12 is mutated from glycine (G) to R, K, A, H, M, L or S, or the 33rd position of the light chain of h1B12 is mutated from asparagine (N) to R.
  • isolated antibodies refer to antibodies that are substantially free of other antibodies with different antigenic specificities, for example, an isolated antibody that specifically binds to a GPC3 protein is substantially free of antibodies that specifically bind to antigens other than GPC3.
  • an isolated antibody that specifically binds to a human GPC3 protein may have cross-reactivity with other antigens (e.g., GPC3 proteins from other species).
  • an isolated antibody may be substantially free of other cellular material and/or chemical substances.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • mouse antibody is intended to include antibodies with variable regions, wherein the framework region and CDR regions are all derived from the immunoglobulin sequences of mouse germline. In addition, if the antibody comprises a constant region, the constant region is also derived from the immunoglobulin sequences of mouse germline.
  • Mouse antibodies of the present invention can include amino acid residues that are not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo.
  • the term “mouse antibody” is not intended to include antibodies whose CDR sequences from another mammalian germline have been transplanted to the mouse framework sequences.
  • chimeric antibody refers to an antibody made by combining genetic material from a non-human source with genetic material from a human. Or more generally, a chimeric antibody is an antibody that has genetic material from one species and genetic material from another species.
  • humanized antibody refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.
  • antibody-drug conjugate refers to the use of the characteristics of antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby achieving the precise delivery of anti-tumor therapeutic agents (such as cytotoxins or cytostatics, radioactive isotopes, small molecule chemotherapeutics, etc.) to tumor target cells, causing intracellular accumulation and release, to achieve the purpose of accurately killing tumors.
  • ADC is also considered to be the most promising anti-tumor drug because of its appropriate molecular weight, high stability, strong targeting, and low toxic side effects.
  • bispecific antibodies can also be coupled to therapeutic agents.
  • the portion coupled to the antibody or bispecific antibody of the present invention to form an antibody conjugate is a cytotoxin, which refers to a cytotoxin that inhibits or prevents cell function and/or causes cell death.
  • the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians and reptiles, although mammals such as non-human primates and rodents are preferred.
  • therapeutically effective amount refers to an amount of the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer) and/or reduce the severity of the disease or condition.
  • a therapeutically effective amount should be understood in the context of the condition being treated, wherein those skilled in the art can readily identify the actual effective amount.
  • the antibodies of the present invention are monoclonal antibodies characterized structurally and chemically as described below and in the following examples.
  • the amino acid sequence ID numbers of the heavy chain/light chain variable regions of the antibodies are summarized in Table 1.
  • Some antibodies have the same VH or VL, for example, hYP7 and its mutant hYP7HM have the same light chain (hYP7-L), and h1B12 and its mutants h1B12-G34R, h1B12-G34K, and h1B12-G34A have the same heavy chain (h1B12-H).
  • the heavy chain constant region of the antibody can be a human IgG1 heavy chain constant region having, for example, an amino acid sequence shown in SEQ ID NO: 54
  • the light chain constant region can be a human immunoglobulin kappa light chain constant region having, for example, an amino acid sequence shown in SEQ ID NO: 55.
  • the heavy chain variable region CDR and light chain variable region CDR in Table 1 are defined by Kabat, Chothia, IMGT, AbM or Contact numbering system/method. However, as is well known in the art, CDR regions may also be based on other heavy chain/light chain variable region sequences such as the CDR regions of the antibodies of the present invention defined by different numbering systems are shown in Table 2.
  • FIG. 1 Schematic diagram of bispecific antibody (heavy chain) construction.
  • the heavy chain (1B12-VH) of the h1B12 antibody is connected to different scFV forms of the second antibody (such as hYP7, h2D8) through two GGGGS linkers.
  • the linker between the VH and VL variable regions of the second antibody can be 3/4 GGGGS, YP7-HM refers to YP7-VH containing the A68G mutation, and IgG1-CH refers to the heavy chain constant region of human IgG1.
  • Figure 2 shows the ELISA binding curves of the bispecific antibody and the humanized monoclonal antibody to the human GPC3 protein. Each antibody has a high affinity to human GPC3 (EC50 is at the sub-nM level).
  • Figures 3A and 3B show the binding of bispecific antibodies and humanized monoclonal antibodies to human liver cancer cells HepG2 or Hep3B.
  • bsDH series bispecific antibodies based on h1B12+h2D8 or each monoclonal antibody
  • bs-FH series bispecific antibodies based on h1B12+hYP7 have higher cell affinity (EC50 values for HepG2 cells and Hep3B are both around 2nM).
  • Figures 4A and 4B show the internalization of bispecific antibodies and humanized monoclonal antibodies in human liver cancer cells HepG2 or Hep3B. Compared with the bsDH series of bispecific antibodies (based on h1B12+h2D8) or each monoclonal antibody, the bs-FH series of bispecific antibodies (based on h1B12+hYP7) showed better antibody internalization.
  • Figures 5A and 5B show the binding of antibody-drug conjugates to human liver cancer cells Hep3B or Huh-7. Compared with monoclonal antibody-drug conjugates, bispecific antibody-drug conjugates showed higher cell affinity in both GPC3 antigen high-expressing cell lines (Hep3B) and low-expressing cell lines (Huh-7).
  • Figures 6A-6D show the killing effect of antibody-drug conjugates on human liver cancer cells HepG2, Hep3B or Huh-7. At high concentrations ( Figures 6A-6C, 66.7 nM) and low concentrations ( Figure 6D, 1 nM), bispecific antibody-drug conjugates showed better killing effects on tumor cells than monoclonal antibody-drug conjugates.
  • Figures 7A and 7B show that the unconjugated bispecific antibody and monoclonal antibody have no direct killing effect on human liver cancer cells HepG2 ( Figure 7A) or Hep3B ( Figure 7B).
  • FIG8A shows the ELISA binding curves of humanized mAbs of 1B12 and its variants to human GPC3 protein.
  • Figures 8B, 8C and 8D show the binding of humanized mAbs of 1B12 and its variants to human liver cancer cells HepG2, Hep3B or Huh-7.
  • Figure 9 shows the internalization of humanized mAb 1B12 in human liver cancer cells Hep3B.
  • mice were immunized according to the method described in E. Harlow, D. Lane, Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.
  • the amino acid residues Gln from position 25 to position 559 of the full-length extracellular domain of human GPC3 protein were mutated.
  • the BALB/c mice were immunized with the amino acid residue His as the immunogen (Antigen 1), and the serum titer was tested after the second and third immunizations. The two mice showing the highest antibody titer were electrofused.
  • Antigen 1 immunogen or His polypeptide antigen from amino acid residue Ser 359 to amino acid residue 559 of the extracellular C-terminal domain (Antigen 7), was used to determine the antiserum titer and screen hybridoma cells secreting antigen-specific antibodies.
  • mice of mouse myeloma cell line (SP2/0-Ag14, ATCC#CRL-1581) were cultured to the logarithmic growth phase.
  • SP2/0-Ag14, ATCC#CRL-1581 mouse myeloma cell line
  • ATCC#CRL-1581 mouse myeloma cell line
  • the immunized mice were killed, the spleens of the immunized mice were removed under sterile conditions, and spleen cell suspensions were prepared.
  • the spleen B cells and mouse myeloma cells in the logarithmic growth phase were fused using PEG chemistry, and the fused cells were continuously cultured.
  • the fused hybridoma cells were then plated into 96-well plates and cultured with DMEM medium containing 20% FCS/HAT. Surviving hybridoma clones can usually be observed under a microscope after 7 to 10 days. Two weeks after cell plating, the culture supernatant of hybridomas in each well was collected, and the hybridoma expressing anti-GPC3 antibodies was screened by ELISA method using recombinant human GPC3 protein (Antigen 1 and Antigen 7). The specific screening steps are as follows: recombinant human GPC3 protein (Antigen 1 or Antigen 7, 2 ⁇ g/mL) was coated in a 96-well plate, 50 ⁇ L/well, and incubated overnight at 4°C.
  • each mouse monoclonal antibody has a high affinity to the GPC3 protein, most of which recognize the C-terminal domain Antigen7 of the GPC3 protein, while m2G12 recognizes the domain outside the C-terminal.
  • the ELISA method was used to detect the recognition of GPC3 proteins of different species by each antibody.
  • Cynomolgus GPC3 (ACRO Biosystems) and Mouse GPC3 (ACRO Biosystems) were coated in 96-well plates at a concentration of 2 ⁇ g/mL, 50 ⁇ L/well, and the detection was carried out according to the above ELISA implementation method. The results are shown in Table 4.
  • competitive ELISA was used to determine whether each candidate antibody recognized the same epitope as the GC33 antibody.
  • the specific steps are: dilute each antibody to 2 ⁇ g/mL, add to a 96-well ELISA plate, 100 ⁇ L/well, and coat overnight at 4°C. Wash the plate three times with PBST, add blocking solution (PBS+1% BSA), 200 ⁇ L/well, and block at 37°C for 1h. Add 2 ⁇ g/mL Insert 1 antigen to each well, 100 ⁇ L/well, incubate at 37°C for 1h, and wash the plate three times with PBST.
  • Hybridoma antibody variable region genes were fished out by 5’RLM-RACE.
  • total RNA of each candidate hybridoma was extracted, and the samples were reverse transcribed using FirstChoice RLM-RACE Kit (Thermo) to obtain cDNA, and PCR was performed to amplify the heavy chain and light chain variable region genes.
  • the PCR products were cloned into a vector for sequencing, and the antibody sequences of m1B12, m2D8, m2G12, m1B8, m1H4, m36C12, and m37F4 were obtained, as shown in Table 6.
  • Each mouse anti-GPC3 monoclonal antibody was selected for humanization and further research.
  • the mouse monoclonal antibody was humanized using a mature CDR transplantation method.
  • the light chain variable region and heavy chain variable region sequences of each mouse monoclonal antibody were BLAST-aligned with the human immunoglobulin gene database to select the receptor framework region for humanization of each mouse antibody.
  • the human germline IGVH and IGVK with the highest homology to each mouse antibody were selected as the receptor framework for humanization.
  • the CDR of each mouse antibody heavy chain variable region or light chain variable region was transplanted into the selected human germline framework region, and the amino acid residues in the framework region were back-mutated to obtain more candidate heavy chain variable regions and/or light chain variable regions of humanized antibodies.
  • the heavy chain variable region and light chain variable region of each antibody after humanization, as well as the mutation site design are summarized in Table 7.
  • the nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was connected to the secretion signal peptide (SP) coding sequence at the front end and the termination codon TAG at the back end, and then gene synthesis was performed and cloned into the pUC57-GW-kan vector (Suzhou Jinweizhi Biotechnology Co., Ltd.), and inserted into the downstream of the CMV promoter of the pCDNA3.1 vector by the In-fusion cloning method.
  • SP secretion signal peptide
  • the human immunoglobulin kappa light chain constant region (IgG-CK) coding sequence was added with a signal peptide (SP) coding sequence at the front end and a termination codon TAG at the rear end, and after gene synthesis, it was inserted downstream of the CMV promoter of the pCDNA3.1 vector to obtain the light chain constant region vector pCDNA3.1-CK.
  • SP signal peptide
  • each humanized monoclonal antibody expression vector the coding sequences of the heavy chain variable region (VH) and light chain variable region (VL) were gene synthesized and inserted between the SP and constant region of the pCDNA3.1-IgG1 or pCDNA3.1-CK vector according to the above-mentioned In-fusion cloning method to obtain the heavy chain expression vector and light chain expression vector of the monoclonal antibody.
  • the heavy chain variable region nucleotide sequence of the second antibody and the light chain variable region nucleotide sequence are connected through the (GGGGS)3 or (GGGGS)4 linker coding sequence to obtain the scFV nucleotide sequence and gene synthesis, and then the scFV coding sequence is inserted between the SP and VH of the above monoclonal antibody heavy chain expression vector to obtain the heavy chain expression vector of the bispecific antibody.
  • Construction of heavy chain vector of bispecific antibody The schematic diagram is shown in Figure 1.
  • mutant expression vector obtained the heavy chain A68G mutation (alanine mutated to glycine) by replacing the 68th residue coding sequence of hYP7-VH from GCC to GGC through gene synthesis.
  • h1B12-VL (G34X) is obtained by replacing the coding sequence of the 34th residue of h1B12-VL from GGC to CGC, AAG, GCC, GTG, ATG, CTG, TAC, CCG, TCC by gene synthesis, obtaining the light chain G34R, G34K, G34A, G34H, G34M, G34L, G34Y, G34P and G34S series mutations (glycine mutated to arginine, lysine, alanine, histidine, methionine, leucine, tyrosine, proline and serine); or replacing the coding sequence of the 33rd residue of h1B12-VL from AAC to CGC and CCG, obtaining the light chain N33R and N33P mutations (asparagine mutated to arginine and proline).
  • the vector construction method is as described above.
  • ExpiCHO-S cells (Thermo) were cultured in complete medium ( High Yield Expression System, containing 30mL/L of Poloxamer 188 solution 10% and 20mL/L of L-glutamine 200mM, Mirus), subculture 24 hours before transfection to ensure that the cell density is 4x106 cells/mL on the second day, and dilute to 2x106 cells/mL before transfection.
  • the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, filtered with a 0.22 ⁇ m filter (Milipore), and the antibody was purified by protein A affinity chromatography. Briefly, a HiTrap Mabselect suRe prepacked column (Cytiva) was equilibrated with 20 mM PB + 0.15 M NaCl buffer with 5 to 10 column volumes, and the filtered supernatant was loaded using an AKTA Avant 150 chromatography system (Cytiva).
  • the purification column was then eluted with 3 column volumes of 20 mM PB + 0.15 M NaCl buffer, 1 column volume of 20 mM PB + 1 M NaCl buffer, and then washed with 20 mM PB until the baseline was stable. Finally, the antibody was eluted with 20 mM citric acid (adjust pH to 3.0 with 20 mM sodium citrate) and the peak 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0), placed in a 1.5 mL tube, and frozen at -80 °C for later use.
  • neutralization buffer (1 M Tris-HCl, pH 9.0
  • each antibody to human GPC3 protein was determined by ELISA method.
  • the specific operation was as follows: recombinant human GPC3 protein (Antigen 1, 1 ⁇ g/mL) was coated in a 96-well plate at 100 ⁇ L/well and incubated at 4°C overnight. Afterwards, the plates were blocked with PBST containing 1% BSA (containing 0.05% Tween-20) at 37°C for 2 hours (200 ⁇ L/well), washed three times with PBST, and each humanized monoclonal antibody/bispecific antibody was first serially diluted with PBST containing 1% BSA and added to a 96-well plate (100 ⁇ L/well).
  • the working concentrations of the bispecific antibodies bsFH1-bsFH6, bsDH1 and bsDH2 were: 20 nM, 10 nM, 10 nM and then diluted in multiples of 4 for 8 points; the working concentrations of the humanized monoclonal antibodies h1B12, h2D8, hYP7HM, h1B12-N33P-G34P, h1B12-G34Y and GC33 were: 30 nM, 15 nM, and then diluted in multiples of 4 for 8 points. Incubated at 37°C for 1 hour, washed three times with PBST.
  • FIG2 and FIG8A except for h1B12-N33P-G34P-VL and h1B12-G34Y mAbs, the other humanized mAbs and bispecific antibodies all have high affinity (sub-nM level) to the GPC3 protein.
  • Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2, Hep3B or Huh-7 cells.
  • the specific operation was as follows: each humanized monoclonal antibody or bispecific antibody was analyzed by FACS. Buffer (PBS + 5% FBS) was used for serial gradient dilution and added into a 96-well U-shaped plate in sequence.
  • the specific operation was as follows: the working concentrations of bsFH1-bsFH6, bsDH1, bsDH2, h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S, h1B12-N33R, h1B12-N33P-G34P, h1B12-G34Y and GC33 were: 300nM, 200nM, 200nM, diluted in sequence by 3 for 8 points; the working concentrations of h2D8 and hYP7HM were: 400nM, 200nM, 200nM, diluted in sequence by 3 for 8 points.
  • HepG2 and Hep3B cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in FACS Buffer. 2 ⁇ 10 5 cells/well were added to a 96-well plate, gently mixed, and incubated on ice for 90min. After centrifugation at 3500rpm for 3min at 4°C, the supernatant was discarded, and then 250 ⁇ L pre-cooled FACS Buffer was added to resuspend the cells, and the centrifugation and washing were repeated 3 times.
  • each bispecific antibody or humanized monoclonal antibody or humanized monoclonal antibody to the cell surface GPC3 antigen is shown in Figure 3A, Figure 8B (HepG2 cells) and Figure 3B, Figure 8C (Hep3B cells), Figure 8D (Huh-7 cells) and Tables 9 and 10.
  • Figures 3A-3B, Figures 8B-8D, Tables 9 and 10 it can be seen that, except for the h1B12-G34Y monoclonal antibody, the other monoclonal antibodies or bispecific antibodies of the present invention have good affinity for HepG2, Hep3B or Huh-7 cells.
  • each humanized monoclonal antibody or bispecific antibody was diluted to 50nM with FACS Buffer and added to a 96-well U-shaped plate at 50 ⁇ L/well.
  • the antibody and cell binding was carried out in the manner of Example 5.
  • Each antibody and cell were incubated for different times: the sample incubated on ice was set as the background 0h, and the remaining samples were incubated at 37°C for 1h, 2h, and 4h, respectively, and immediately transferred to ice after the incubation was completed.
  • each humanized monoclonal antibody or bispecific antibody was diluted to 200nM with FACS Buffer and added to a 96-well U-shaped plate at 50 ⁇ L/well.
  • the antibody and cell binding was carried out in the manner of Example 5.
  • Each antibody and cell were incubated for different times: the sample incubated on ice was set as the background 0h, and the remaining samples were incubated at 37°C for 1h, 2h, and 4h, respectively, and immediately transferred to ice after the incubation was completed.
  • the cell internalization efficiency of the bispecific antibody or humanized monoclonal antibody of Example 2 or 3 was calculated by the formula: (1-antibody 37°C MFI value/antibody 4°C MFI value) ⁇ 100%.
  • the specific results are shown in Figure 9 (Hep3B cells). According to Figure 9, it can be seen that the monoclonal antibody of the present invention has a very good internalization efficiency for Hep3B cells, and the internalization rate of the monoclonal antibody of the present invention is also significantly better than that of GC33.
  • ADC products were obtained.
  • SEC size exclusion chromatography
  • DAR drug-antibody coupling ratio
  • HIC hydrophobic interaction chromatography
  • Antibody-drug conjugates for killing tumor cells are provided.
  • HepG2, Hep3B, and Huh-7 liver cancer cells were cultured using 10% FBS (Gibco) + DMEM medium (Corning). When the cell confluence reached more than 75%, they were digested with trypsin (0.25% Trypsin-EDTA) and counted. 1.5 ⁇ 10 4 cells/mL and 160 ⁇ L/well (2400 cells/well) were plated on 96-well plates, respectively, and placed in a 37°C, 5% CO 2 incubator.
  • Cell Counting Kit-8 (Dojindo) was used to determine the killing effect of each anti-GPC3 antibody on HepG2 and Hep3B cells.
  • the specific operation was as follows: using 10% FBS + DMEM culture medium, each anti-GPC3 antibody was diluted to 400nM, 80nM, 16nM, 3.2nM, 0.64nM, and 0.13nM, and HepG2 and Hep3B cells were added to a 96-well plate at 1 ⁇ 10 4 cells/mL and 100 ⁇ L/well (10000cells/well).

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Abstract

本发明提供了一种单克隆抗体（特别是小鼠、嵌合的或人源化的单克隆抗体）/双特异性抗体或其抗原结合片段，该抗体或其抗原结合片段以高亲和力和功能活性特异性结合至人GPC3。本发明，还提供了编码所述抗体的核苷酸分子，用于表达所述抗体的表达载体和宿主细胞，以及所述抗体的制备方法。此外，本发明还提供了包含所述抗体的药物组合物，以及所述抗体用于制备治疗癌症的药物用途。

Description

抗GPC3单克隆抗体/双特异性抗体或其抗原结合片段及其用途 技术领域
本发明涉及一种单克隆抗体(特别是小鼠、嵌合的或人源化的单克隆抗体)/双特异性抗体或其抗原结合片段，该抗体或其抗原结合片段以高亲和力和功能活性特异性结合至人GPC3。本发明还提供了编码该抗体或抗原结合片段的核酸分子，用于表达所述抗体或抗原结合片段的表达载体、宿主细胞和方法。本发明进一步提供了包含所述抗体或其抗原结合片段的药物偶联物、单克隆抗体、双特异性分子和药物组合物，以及使用本发明的抗GPC3单克隆抗体/双特异性抗体或其抗原结合片段的诊断和治疗方法。
背景技术
GPC3是一种硫酸乙酰肝素蛋白聚糖，表达于多种恶性细胞的表面，例如肝细胞癌(HCC)细胞。Glypican-3通过糖基-磷脂酰肌醇锚(GPI)与细胞表面相连。GPC3已被证明在超过70％的肝细胞癌活检组织中高度表达，但在邻近的非肿瘤组织中却没有。GPC3阳性HCC患者的无病生存率明显低于GPC3阴性HCC患者。
人们已发现某种类型的结合于GPC3的抗体通过抗体依赖性细胞介导的细胞毒性(ADCC)活性以及补体-依赖的细胞毒性(CDC)活性具有细胞生长-抑制活性(国际专利申请WO 2003/000883)。此外，已表明GPC3在体内裂解并以GPC3的分泌形式分泌到血液中，且利用能够检测分泌形式GPC3的抗体可以进行肿瘤诊断(国际专利申请WO2004/022739，WO 03/100429和WO 2004/018667)。例如：
专利CN1842540B公开了一种抗GPC3抗体，与传统抗体相比具有较高ADCC活性和CDC活性，但是其对GPC3表位的结合能力仍然较弱；
专利CN104520331B公开了一种特异性针对磷脂酰肌醇蛋白聚糖3(GPC3)的高亲和力抗体，尽管其对GPC3表位的结合能力有所提升，但是其细胞毒性仍然有待提高；
尽管如此，由于原发性或者继发性肿瘤耐药性以及肿瘤异质性，依靠单一表位抗体以及抗体自身的生理作用来达到抑制肿瘤生长的已经越来越困难，而同时识别GPC3双表位的双特异性抗体，可以对肿瘤细胞有更强的结合能力从而可以开发更强杀伤力的药物组合物。
抗体偶联药物(antibody-drug conjugate，ADC)是一种新型的靶向性药物治疗方法，是将抗体与具有强细胞毒性的小分子化药偶联而成，兼具小分子药物强大的杀伤力和单抗高度的靶向性，因而成为肿瘤靶向治疗的研究和发展热点。ADC一般包括采用一定方式连接的三个部分：抗体或抗体类配体、连接子(Linker)和小分子化药。ADC的靶向性来自其中抗体部分，毒性主要来自小分子化药，抗体部分也可以带有毒性。抗体部分与肿瘤细胞表面抗原结合后，被内吞进入细胞，之后ADC药物会在溶酶体中分解，释放出有活性的化药毒物，破坏DNA或阻止肿瘤细胞分裂，最终杀死肿瘤细胞。ADC相对其他治疗方式具有以下特点：治疗效力强；肿瘤细胞特异度高，误杀率低，治疗安全窗口更大；免疫原性弱，不容易产生抗药性；血清中循环时间长(短于裸抗)；对非靶点细胞毒性弱。
因此，目前，亟需开发一种既对GPC3表位具有很强的结合能力，又具有良好细胞毒性的抗GPC3单克隆/双特异性抗体或其抗原结合片段以及包含该抗体或抗原结合片段的ADC药物。
发明内容
本发明提供了一种抗GPC3单克隆/双特异性抗体或其抗原结合片段，例如小鼠、人、嵌合的或人源化的单克隆抗体或其抗原结合片段，及含有两种抗原结合结构域的双特异性抗体，所述的抗体或其抗原结合片段结合至GPC3蛋白，所述的抗体或其抗原结合片段具有良好的GPC3蛋白结合亲和力。
一个方面，本发明提供一种抗GPC3单克隆抗体或其抗原结合片段，所述的单克隆抗体或其抗原结合片段结合至GPC3蛋白。
一个方面，本发明提供一种抗GPC3单克隆抗体或其抗原结合片段，其特征在于：所述抗GPC3单克隆抗体或其抗原结合片段包含重链可变区(VH)和链可变区(VL)；
所述重链可变区(VH)包含CDR区，所述CDR区包含与氨基酸序列SEQ ID Nos：14或SEQ ID Nos：16中任一条相同的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，所述CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义；
所述轻链可变区(VL)包含CDR区，所述CDR区包含与氨基酸序列SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60任一条相同的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，所述CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义。
在一方面，作为本发明的优选技术方案，所述重链可变区(VH)的CDR区选自：
1)SEQ ID Nos：14的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
2)SEQ ID Nos：16的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
所述轻链可变区(VL)的CDR区选自：
1)SEQ ID Nos：15的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
2)SEQ ID Nos：17的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
3)SEQ ID Nos：44的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
4)SEQ ID Nos：45的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
5)SEQ ID Nos：46的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
6)SEQ ID Nos：56的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
7)SEQ ID Nos：57的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
8)SEQ ID Nos：58的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
9)SEQ ID Nos：59的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
10)SEQ ID Nos：60的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
在一方面，作为本发明的优选技术方案，所述重链可变区(VH)的CDR区选自：
1)SEQ ID Nos：14的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
所述轻链可变区(VL)的CDR区选自：
5)SEQ ID Nos：15的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
6)SEQ ID Nos：44的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
7)SEQ ID Nos：45的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
8)SEQ ID Nos：46的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
在一方面，作为本发明的优选技术方案，所述重链可变区(VH)包含与氨基酸序列SEQ ID Nos：14或SEQ ID Nos：16相同的序列；
所述轻链可变区(AVL)包含与氨基酸序列SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60的相同的序列。
在一方面，作为本发明的优选技术方案，所述抗GPC3单克隆抗体或其抗原结合片段还包括重链(H)和轻链(L)，所述重链(H)包含与氨基酸序列SEQ ID Nos：61或SEQ ID Nos：71相同的序列；
所述轻链(L)包含与SEQ ID Nos：62、SEQ ID Nos：63、SEQ ID Nos：64、SEQ ID Nos：65、SEQ ID Nos：66、SEQ ID Nos：67、SEQ ID Nos：68、SEQ ID Nos：69或SEQ ID Nos：70的相同的序列。
在一方面，作为本发明的优选技术方案，所述抗GPC3单克隆抗体或其抗原结合片段还包括重链(H)和轻链(L)，所述重链(H)包含与氨基酸序列SEQ ID Nos：61相同的序列；
所述轻链(L)包含与SEQ ID Nos：62、SEQ ID Nos：63、SEQ ID Nos：64或SEQ ID Nos：65相同的序列。
在另一方面，本发明还提供一种抗GPC3双特异性抗体(例如，小鼠、嵌合的或人源化的抗体)或其抗原结合片段，所述抗体或其抗原结合片段结合至GPC3蛋白，所述抗体或抗原结合片段含有第一GPC3抗原结合结构域和第二GPC3抗原结合结构域。
进一步地，所述第一GPC3抗原结合结构域和第二GPC3抗原结合结构域均可包含以下各项或由其组成：(a)重链可变区(VH)和轻链可变区(VL)；(b)单链抗体(scFv)；(c)双体抗体；(d)小抗体；(e)F(ab’)2；(f)F(ab)。或该领 域内其他已知的抗体衍生物等。
进一步地，所述第一GPC3抗原结合结构域和第二GPC3抗原结合结构域均可为以下各项或由其组成：动物源抗体、人源化抗体、全人抗体、嵌合抗体、或亲和力优化的抗体。更优选为人源化抗体、人动物嵌合抗体(如人鼠嵌合抗体)，更优选为全人抗体。所述抗体可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD抗体或其他亚型的抗体中的任意一种或几种。优选的是IgG1、IgG2或IgG4亚型。更优选的，所述IgG1抗体为hIgG1抗体或其变体，在一些实施例中所述的hIgG1的氨基酸序列如SEQ ID NO：30所示，或与SEQ ID NO：30具有至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性；
进一步地，所述第一GPC3抗原结合结构域和第二GPC3抗原结合结构域特异性结合至GPC3的不同表位；
进一步地，所述双特异性抗GPC3抗体中，第一GPC3抗原结合结构域和第二GPC3抗原结合结构域的连接关系为如下(a1)或(a2)：
(a1)所述第一GPC3抗原结合结构域的重链可变区和轻链可变区通过接头L1连接形成scFv，所述scFv进一步通过接头L2连接至所述第二GPC3抗原结合结构域的重链或轻链；
(a2)所述第二GPC3抗原结合结构域的重链可变区和轻链可变区通过接头L1连接形成scFv，所述scFv进一步通过接头L2连接至所述第一GPC3抗原结合结构域的重链或轻链；
所述接头L1和L2均独立地选自(GGGGS)n，其中，n为1、2、3、4、5或6的整数；
优选地，所述scFv进一步通过接头L2连接至所述第一或第二GPC3抗原结合结构域的重链或轻链末端；
更优选地，所述重链或轻链末端选自重链或轻链C端或N端。
再进一步地，所述抗GPC3双特异性抗体包含第一多肽链和第二多肽链；
其中，所述第一多肽链的结构可选自如下(b1)-(b8)中任一：
(b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH1]-[Fcx]；
(b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH1]-[Fcx]；
(b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH1]-[Fcx]；
(b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH1]-[Fcx]；
(b5)[BVH]-[CH1]-[Fcx]-[L2]-[AVH]-[L1]-[AVL]；
(b6)[BVH]-[CH1]-[Fcx]-[L2]-[AVL]-[L1]-[AVH]；
(b7)[AVH]-[CH1]-[Fcx]-[L2]-[BVH]-[L1]-[BVL]；
(b8)[AVH]-[CH1]-[Fcx]-[L2]-[BVL]-[L1]-[BVH]；
其中，[L1]、[L2]独立地表示接头，所述接头选自(GGGGS)n，其中，n为1、2、3、4、5或6的整数；所述CH1为抗体重链恒定区1；所述[Fcx]表示Fc结构域；
[AVH]和[AVL]分别表示所述第一GPC3抗原结合结构域的重链可变区(AVH)和轻链可变区(AVL)；
[BVH]和[BVL]分别表示所述第二GPC3抗原结合结构域的重链可变区(BVH)和轻链可变区(BVL)；
[Fcx]表示Fc结构域，包含CH2和CH3；
所述第二多肽链的结构可选自如下(c1)或(c2)：
(c1)[AVL]-[CL]；
(c2)[BVL]-[CL]；
所述[CL]为抗体轻链恒定区；
[AVL]-[CL]表示所述第一GPC3抗原结合结构域的轻链可变区(AVL)和轻链恒定区(CL)；
[BVL]-[CL]表示所述第二GPC3抗原结合结构域的轻链可变区(BVL)和轻链恒定区(CL)。
所述第一多肽链(重链)和所述第二多肽链(轻链)之间通过二硫键连接。
进一步地，所述第一重链可变区(AVH)的CDR区选自SEQ ID Nos：14、16、34、49或51中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
进一步地，所述第一轻链可变区(AVL)的CDR区选自SEQ ID Nos：15、17、35、44、45、46、50、52、56、57、58、59或60中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
进一步地，所述第二重链可变区(BVH)的CDR区选自SEQ ID Nos：1、3、5、6、8、12、18、20、22、24、26、28、30、32或47中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
进一步地，所述第二轻链可变区(BVL)的CDR区选自SEQ ID Nos：2、4、7、9、10、11、13、19、21、23、25、27、29、31、33或48中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的。
进一步地，所述第一重链可变区(AVH)的CDR区选自：
1)SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104 位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
2)SEQ ID Nos：16(m1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
进一步地，所述第一轻链可变区(AVL)的CDR区选自：
1)SEQ ID Nos：15(h1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
2)SEQ ID Nos：17(m1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
3)SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
4)SEQ ID Nos：45(h1B12-G34K)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
5)SEQ ID Nos：46(h1B12-G34A)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
6)SEQ ID Nos：56的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
7)SEQ ID Nos：57的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
8)SEQ ID Nos：58的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
9)SEQ ID Nos：59的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
10)SEQ ID Nos：60的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
进一步地，所述第二重链可变区(BVH)的CDR区选自：
1)SEQ ID Nos：1(YP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
2)SEQ ID Nos：3(hYP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
3)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
4)SEQ ID Nos：6(YP8)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
5)SEQ ID Nos：8(YP9)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
6)SEQ ID Nos：12(YP9.1)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
7)SEQ ID Nos：18(h2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
8)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
进一步地，所述第二轻链可变区(BVL)的CDR区选自：
1)SEQ ID Nos：2(YP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
2)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
3)SEQ ID Nos：7(YP8)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
4)SEQ ID Nos：9(YP9克隆9)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
5)SEQ ID Nos：10(YP9克隆10)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
6)SEQ ID Nos：11(YP9克隆1)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
7)SEQ ID Nos：13(YP9.1)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
8)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
9)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
优选地，所述第一重链可变区(AVH)的CDR区选自：SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组。
所述第一轻链可变区(AVL)的CDR区选自：
1)SEQ ID Nos：15(h1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
2)SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
3)SEQ ID Nos：45(h1B12-G34K)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
4)SEQ ID Nos：46(h1B12-G34A)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
优选地，所述第二重链可变区(BVH)的CDR区选自：
1)SEQ ID Nos：1(YP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
2)SEQ ID Nos：3(hYP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
3)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
4)SEQ ID Nos：18(h2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
5)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
优选地，所述第二轻链可变区(BVL)的CDR区选自：
1)SEQ ID Nos：2(YP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
2)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组
3)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
4)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
优选地，所述第一重链可变区(AVH)的CDR区选自：SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104 位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
所述第一轻链可变区(AVL)的CDR区选自：SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
优选地，所述第二重链链可变区(BVH)的CDR区选自：
1)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
2)SEQ ID Nos：18(h2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
3)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
所述第二轻链可变区(BVL)的CDR区选自：
1)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
2)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
3)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
优选地，所述第一重链可变区(AVH)的CDR区选自：h1B12或m1B12中任一条的重链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
优选地，所述第一轻链可变区(AVL)的CDR区选自h1B12、h1B12-G34R、h1B12-G34K、h1B12-G34A或m1B12中任一条的轻链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
优选地，所述第二重链可变区(BVH)的CDR区选自：YP7、hYP7、hYP7HM、YP8、YP9、YP9.1、h2D8或m2D8中任一条的重链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
优选地，所述第二轻链可变区(BVL)的CDR区选自：YP7、hYP7、YP8、YP9、YP9.1、h2D8或m2D8中任一条的轻链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的。
优选地，所述第一重链可变区(AVH)选自SEQ ID Nos：14、16、34、49或51中任一条；
优选地，所述第一轻链可变区(AVL)区选自SEQ ID Nos：15、17、35、44、45、46、50、52、56、57、58、59或60中任一条；
优选地，所述第二重链可变区(BVH)选自SEQ ID Nos：1、3、5、6、8、12、18、20、22、24、26、28、30、32或47中任一条；
优选地，所述第二轻链可变区(BVL)选自SEQ ID Nos：2、4、7、9、10、11、13、19、21、23、25、27、29、31、33或48中任一条。
优选地，所述抗GPC3双特异性抗体的第一多肽链包含：SEQ ID Nos：36；SEQ ID Nos：37；SEQ ID Nos：38；SEQ ID Nos：39；SEQ ID Nos：40；SEQ ID Nos：41；SEQ ID Nos：42或SEQ ID Nos：43中的任一条。
优选地，所述双特异性抗GPC3抗体的第二多肽链由轻链可变区(VL)和轻链恒定区(CL)组成，其中VL包含：SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60中的任一条，CL包含：SEQ ID Nos：55；
更优选地，所述双特异性抗GPC3抗体的第二多肽链由轻链可变区(VL)和轻链恒定区(CL)组成，其中VL包含：SEQ ID Nos：15；SEQ ID Nos：17；SEQ ID Nos：44；SEQ ID Nos：45或SEQ ID Nos：46中的任一条，CL包含：SEQ ID Nos：55；
另一方面，本发明还提供一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包含本发明所述的抗GPC3单克隆抗体或其抗原结合片段或者抗GPC3双特异性抗体或其抗原结合片段。
另一方面，本发明还提供一种多核苷酸，其特征在于，所述多核苷酸编码本发明所述的抗GPC3单克隆抗体或其抗原结合片段或者抗GPC3双特异性抗体或其抗原结合片段。本发明的多核苷酸可以是，例如DNA或RNA， 并可包含或可不包含内含子序列。在优选实施方案中，所述多核苷酸为cDNA分子。
另一方面，本发明还提供一种表达载体，其特征在于，所述表达载体包含本发明所述的多核苷酸。所述的表达载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
另一方面，本发明还提供一种宿主细胞，其特征在于，所述宿主细胞包含本发明所述的多核苷酸或本发明所述的表达载体。所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NSO细胞或其它哺乳动物细胞，优选为CHO细胞。
另一方面，本发明还提供一种抗体-药物偶联物，其特征在于，其包含本发明所述的抗GPC3单克隆抗体或其抗原结合片段或者本发明所述的抗GPC3双特异性抗体或其抗原结合片段以及药物或毒素。
另一方面，作为本发明的一种优选技术方案，所述药物或毒素选自：SN-38、MMAE、PBD dimer、DX-8951(DXd)或DUBA中的一种或多种。
所述抗GPC3双特异性抗体可按照包括如下步骤的方法制备获得：
1)构建表达载体，所述表达载体是将编码如前所述的任一抗GPC3双特异性抗体的核苷酸连接到基础载体上得到的；
2)将步骤1)构建的表达载体转染或转化至宿主细胞中，并培养宿主细胞；
3)分离、纯化抗GPC3双特异性抗体。
本发明所述的抗GPC3双特异性抗体或其抗原结合片段的DNA分子的序列可以采用常规技术获得，例如利用杂交瘤PCR扩增或噬菌体展示文库筛选等。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体(例如scFV)。
一旦获得了有关的序列，就可以将其克隆入载体，再转入宿主细菌，然后通过常规方法从宿主细菌中提取得到有关载体。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如细菌)，或是低等真核细胞(如酵母)，或是高等真核细胞(如哺乳动物细胞)。优选的动物细胞包括(但并不限于)：CHO、HEK-293细胞。
本发明的抗GPC3双特异性抗体或其抗原结合片段可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞，然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose亲和层析、离子交换层析、疏水层析、分子筛层析、羟基磷灰石层析、凝胶电泳、透析等常规分离纯化手段及这些方法的结合，纯化得到本发明的抗GPC3双特异性抗体或其抗原结合片段。
作为本发明所述抗GPC3双特异性抗体或其抗原结合片段的制备方法的优选实施方式，所述分离、纯化抗GPC3双特异性抗体或其抗原结合片段的方法为蛋白A亲和层析法、阳离子交换法或阴离子交换法。
所得单克隆抗体或双特异性抗体可用常规手段来鉴定。例如，抗体的结合特异性可用免疫沉淀或体外结合试验，如酶联免疫吸附分析(ELISA)或放射性免疫分析(RIA)来测定。抗体的结合亲和力例如可用Munson等人，Anal.Biochem.，107：220(1980)的Scatchard分析来测定。
本发明提供了一种免疫偶联物，所述免疫偶联物含有：
1)如本发明所述的抗GPC3双特异性抗体、单克隆抗体或其抗原结合片段；和
2)选自下组的偶联部分：药物、毒素、可检测标记物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
作为优选，所述的放射性核素包括：
(a)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或
(b)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
作为优选，所述偶联部分为药物或毒素。
作为优选，所述的药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
作为优选，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、DNA 烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱，或其组合。
作为优选，特别有用的细胞毒性药物的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂，典型的细胞毒性药物包括，例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1，4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)，或其组合。
作为优选，所述的毒素选自下组：耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素，或其组合。
作为优选，所述的药物或毒素选自：SN-38(NK012，CAS No.：86639-52-3)、MMAE(Monomethyl auristatin E，CAS No.：474645-27-7)、PBD dimer(SG3199，CAS No.：1595275-71-0)、DX-8951(Exatecan，CAS No.：171335-80-1)或DUBA(duocarmycin-hydroxybenzamide-azaindole)中的一种或多种。
所述抗体可以和所述药物偶联从而形成抗体药物偶联物(ADC)。典型地，ADC包含一种本发明所述的抗GPC3双特异性抗体、单克隆抗体或抗原结合片段，通过接头连接至一种药物或毒素。所述接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，从而使治疗剂从抗体上释放出来。合适的可降解的接头包括，酶降解的接头例如，可以被细胞内溶酶体蛋白酶降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括二肽例如，缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，pH敏感接头(例如pH小于5.5时水解的腙接头)，在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，例如马来酰亚胺类化合物，卤代酰胺，卤代酯，卤代甲基酮，苄基卤代物，乙烯基砜，吡啶基二硫化物，汞衍生物，和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
作为优选，所述接头连接的药物或毒素选自：CL2A-SN-38(CAS No.：1279680-68-0)、mc-vc-PAB-MMAE(CAS No.：646502-53-6)、Tesirine(SG3249，CAS No.：1595275-62-9)、Deruxtecan(CAS No.：1599440-13-7)、Vc-seco-DUBA(SYD985，CAS No.：1345681-58-4)。分子结构如下图所示：
在本发明中，所述抗GPC3双特异性抗体或单克隆抗体通过CL2A接头偶联SN-38。
在本发明中，所述抗GPC3双特异性抗体或单克隆抗体通过mc-VC-PAB接头偶联MMAE。
在本发明中，所述抗GPC3双特异性抗体或单克隆抗体通过maleimide-dPEG8-VA-PABA接头偶联PBD dimer。
在本发明中，所述抗GPC3双特异性抗体或单克隆抗体通过maleimide-GGFG接头偶联DX-8951(DXd)。
在本发明中，所述抗GPC3双特异性抗体或单克隆抗体通过Vc-seco接头偶联DUBA。
在本发明中，所述抗体-药物偶联物是按照包括如下步骤的方法制备获得的：
前述任一所述的抗GPC3双特异性抗体的链间二硫键被还原，产生2n个(如2，4，6，8个)巯基基团；
药物-接头化合物与还原后的抗体巯基交联，生成相应的抗体药物偶联物；
经超滤脱盐进一步纯化得到产物。
在一个方面，本发明还提供了一种药物组合物，所述药物组合物包含本发明所述的抗GPC3单克隆抗体或其抗原结合片段或者抗GPC3双特异性抗体或其抗原结合片段，或抗体-药物偶联物、或溶瘤病毒，以及药学上可接受的载体。当组合物包含一种以上抗体(或其抗原结合片段、或抗体-药物偶联物、或溶瘤病毒)时，可以分批施用抗体(或其抗原结合片段、或抗体-药物偶联物、或溶瘤病毒)。该组合物可以任选地包含一种或多种另外的药物活性成分，例如另一抗体或药物，例如抗肿瘤药物。
药物组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠剂或乳化剂、固体粘合剂、分散或悬浮助剂、增溶剂、着色剂、调味剂、包衣剂、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂或其组合。在如下中教导了选择和使用合适的赋形剂，Gennaro编著，Remington：The Science and Practice of Pharmacy，第20版(Lippincott Williams & Wilkins 2003)，以引用的方式将其公开内容并入本文。
另一方面本发明提供一种编码本发明的抗GPC3双特异性抗体或其抗原结合片段的核苷酸分子。所述核酸分子可以存在于完整细胞或细胞裂解物中，或者是部分纯化的或大致纯的形式。当使用标准技术将核酸同其他细胞组分或其他污染物(如细胞内的其他核酸或蛋白)分开时，该核酸即为“分离的”或“视为大致纯”，所述标准技术包括碱性/SDS裂解法、氯化铯密度梯度离心法(CsCl banding)、柱层析法、琼脂糖凝胶电泳和本领域公知的其他方法。参见F.Ausubel等人编(1987)Current Protocols in Molecular Biology(分子生物学最新方案)GreenePublishing and Wiley Interscience，New York。本发明的核酸可以是，例如DNA或RNA，并可包含或可不包含内含子序列。在优选实施方案中，所述核酸为cDNA分子。
一方面，本发明提供了一种载体，包含本发明所述的核苷酸。所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明另一方面提供了包含上述载体或抗体的宿主细胞；所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NSO细胞或其它哺乳动物细胞，优选为CHO细胞。
本发明公开的抗GPC3双特异性抗体或其抗原结合片段还可用于制备嵌合抗原受体(CAR；也称为嵌合T细胞受体、人造T细胞受体或嵌合免疫受体)。
本发明另一方面提供本发明所述的抗GPC3单克隆抗体或其抗原结合片段或者抗GPC3双特异性抗体或其抗原结合片段、抗体-药物偶联物、核苷酸、载体或宿主细胞在制备自身免疫疾病、病毒感染或癌症的治疗药物或诊断试剂中的应用，所述癌症为肝癌、胃癌、结直肠癌、肺癌或卵巢癌等，或表达GPC3的任何其他类型的癌症。
本发明还提供了一种治疗患有癌症例如肝癌的受试者的方法：选出患有表达GPC3的癌症的受试者，给予所述受试者治疗有效量的抗GPC3双特异性抗体或其抗原结合片段，或包含所述抗体的抗体-药物偶联物。
在另一方面，本发明提供了一种抑制受试者肿瘤生长的方法，该方法包括向受试者给予有效剂量的包含本发明所述的抗GPC3双特异性抗体或其抗原结合片段的药物或其组合物。所述肿瘤可以是实体瘤或非实体瘤，包括但不限于，肝癌、B细胞淋巴瘤、慢性淋巴细胞白血病、多发性骨髓瘤、黑素瘤、结肠腺癌、胰腺癌、结肠癌、胃肠癌、前列腺癌、膀胱癌、肾癌、卵巢癌、子宫颈癌、乳腺癌、肺癌和鼻咽癌。在一些实施方式中，所述方法包括给予本发明的组合物、抗GPC3双特异性抗体或其抗原结合片段、抗体-药物偶联物、或编码抗体或携载抗体的溶瘤病毒，或在受试者体内能够表达的前述相同物质的核酸分子。在一些实施方式中，可以将至少一种额外的抗肿瘤抗体联合本发明的抗GPC3双特异性抗体或其抗原结合片段一起施用，所述至少一种额外的抗肿瘤抗体可以选自抗VISTA抗体、抗PD-1抗体、抗PD-L1抗体、抗LAG-3抗体、抗CD40抗体、抗TIM-3抗体、抗STAT3抗体和/或抗ROR1抗体。在另一个实施方式中，本发明的抗体或其抗原结合片段联合一种化疗剂一起施用，所述的化疗剂可以是一种细胞毒剂，如表柔比星、奥沙利铂和/或5-氟尿嘧啶(5-FU)。本发明的抗体可以是小鼠、人、嵌合的或人源化的抗体。
为了确保可以更容易地理解本发明，首先定义某些术语，在整个详细描述中对附加定义进行了阐述：
术语“GPC3”(也称为磷脂酰肌醇蛋白聚糖3)是硫酸肝素蛋白聚糖的一员，通过糖基磷脂酰肌醇(glycosyl-phosphatidylinositol，GPI)锚定于细胞膜表面。人GPC3基因位于X染色体(Xp26)上并编码70kDa蛋白质，该蛋白质含有580个氨基酸，在Arg358和Ser359之间被弗林蛋白酶样转化酶内切切割，产生40kDa的N末端亚基和30kDa的C末端亚基，C末端亚基上还有两条硫酸乙酰肝素(heparan sulfate，HS)链。
如本文所使用的，“特异性结合至人GPC3”的抗体是指结合至人GPC3蛋白(以及可能来自一种或多种非人类物种的GPC3蛋白)但实质上是不结合至非GPC3蛋白的抗体。
本文所指的术语“抗体”包括完整抗体及其任意抗原结合片段(即，“抗原结合部分”)或其单链。完整抗体是包含通过二硫键链间连接的两条重(H)链和两条轻(L)链的糖蛋白。每条重链由重链可变区(在本文中缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步细分为高 变区(称为互补决定区(CDR))，其间间隔着更为保守的区域，称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)。
术语“双特异性抗体”：是指能够结合两个独立抗原或对同一抗原内不同表位具有结合特异性的抗体分子。例如，在一些实施方案中，双特异性抗体分子的一个抗原结合片段结合GPC3第一抗原结合结构域，另一个抗原结合片段结合GPC3第二抗原结合结构域，这样可以进一步增强与细胞表面GPC3抗原的亲和力。
如本文所使用的，术语抗体的“抗原结合片段”(或简称为“抗体部分”)是指与抗原(例如，GPC3蛋白)特异性结合的抗体的一个或多个片段。已经显示出了抗体的抗原结合功能可以通过全长抗体的片段得以实现。术语抗体的“抗原结合片段”所涵盖的结合片段的实例包括：(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab’)2片段，包含在铰链区通过二硫键连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(v)由VH结构域组成的dAb片段(Ward等，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，包含单个可变域和两个恒定结构域的重链可变区。此外，尽管FV片段的两个结构域VL和VH由分隔开的基因编码，但是它们可以使用重组方法通过接头连接起来，从而使它们成为单一蛋白链，其中，VL区和VH区配对以形成单价分子(称为单链Fv(scFv)；参见例如Bird等，(1988)Science 242：423-426；以及Huston等，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883)。此类单链抗体也包含在术语抗体的“抗原结合片段”范围内。这些抗体片段可通过本领域技术人员已知的常规技术获得，为使用而进行的片段筛选与完整抗体的方法相同。
术语“scFv”：包含免疫球蛋白的轻链可变区和重链可变区的经遗传改造的分子，通过适合的多肽接头连接为基因上融合的单链抗体。
术语“Fc”：Fc区表示抗体分子的Fc片段区域，包含铰链区、CH2和CH3结构域的一部分，并且具有大约50,000Da的分子量。在抗体用木瓜蛋白酶消化时，人IgG重链Fc区是从第225位的苏氨酸到C末端。
术语“IgG1”是指人源或鼠源重链恒定区部分，其中“hIgG1”是指人源重链恒定区部分，所述“IgG1”或“hIgG1”包括CH1、CH2和CH3结构域；
术语“Fab”，包含抗体分子的单价抗原结合片段的片段，其可以通过用木瓜蛋白酶消化完整抗体以产生完整轻链和一条重链的一部分而产生。
术语“Fab′”，可以通过用胃蛋白酶处理完整抗体、之后进行还原以产生完整轻链和重链的一部分而获得的抗体分子片段；每个抗体分子得到两个Fab′片段。
术语“(Fab′)2”，可以通过将完整的抗体用胃蛋白酶处理、但之后不进行还原而得到的抗体片段；F(ab′)2是两个Fab′片段通过两个二硫键连接在一起的二聚体。
术语“Fv”，含有表达为2条链的轻链可变区和重链可变区的基因程片段。
术语“h1B12-G34R、h1B12-G34K、h1B12-G34A、h1B12-G34H、h1B12-G34M、h1B12-G34L、h1B12-G34S或h1B12-N33R”是指h1B12的轻链第34位由甘氨酸(G)突变为R、K、A、H、M、L或S，或者h1B12的轻链第33位由天冬酰胺(N)突变为为R而获得的抗体或其抗原结合片断。
如本文所使用的，“分离的抗体”指实质上不含有其它具有不同抗原特异性抗体的抗体，例如，特异性结合GPC3蛋白的分离抗体实质上不含与GPC3之外的其他抗原特异性结合的抗体。但是，例如，在一些实施例中，特异性地结合人GPC3蛋白的分离的抗体可以与其它抗原(例如来自其它物种的GPC3蛋白)具有交叉反应性。此外，分离的抗体可以实质上不含其它细胞物质和/或化学物质。
如本文所使用的，术语“单克隆抗体”是指具有单一分子组成的抗体分子的制剂。单克隆抗体组成表现出对特定表位的单一结合特异性和亲和力。
如本文所使用的，术语“小鼠抗体”旨在包括具有可变区的抗体，其中，框架区和CDR区均来源于小鼠种系的免疫球蛋白序列。此外，如果抗体包含恒定区，则该恒定区也来源于小鼠种系的免疫球蛋白序列。本发明的小鼠抗体可以包括不是由小鼠种系免疫球蛋白序列编码的氨基酸残基，例如在体外通过随机或定点诱变或在体内通过体细胞突变所引入的突变。然而，如本文所使用的，术语“小鼠抗体”并不旨在包括来自另一哺乳动物种系的CDR序列已被移植到小鼠框架序列上的抗体。
术语“嵌合抗体”是指通过将来自非人来源的遗传物质和来自人类的遗传物质的结合而制成的抗体。或者更通常地，嵌合抗体是具有来自某物种的遗传物质和来自另一物种的遗传物质的抗体。
如本文所使用的，术语“人源化抗体”是指来自非人类物种的抗体，其蛋白质序列已被修饰以增加其与人类天然产生的抗体变体的相似性。
术语“抗体-药物偶联物”是指利用抗体特异性识别肿瘤细胞表面特定抗原的特点，从而实现精准地将抗肿瘤治疗剂(如细胞毒素或细胞抑制剂、放射性同位素、小分子化疗物等)递送到肿瘤靶细胞，发生胞内积蓄并释放，达到精准杀伤肿瘤的目的。ADC也因为其分子量大小合适，稳定性高，靶向性强，毒副作用小被认为是最具潜力的抗肿瘤药物。除单克隆抗体外，双特异性抗体也可以偶联治疗剂。在一些实施方案中，与本发明的抗体或双特异性抗体偶联以形成抗体偶联物的部分是细胞毒素，所述细胞毒素是指抑制或阻止细胞功能和/或引起细 胞破坏的物质，并包括小分子细胞毒素。在一些实施方案中，所述细胞毒素选自SN-38、MMAE、PBD dimer、DX-8951(DXd)或DUBA。
术语“受试者”包括任何人类或非人类的动物。术语“非人类的动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，比如，非人类的灵长类动物、啮齿类动物、家兔、猪、狗、猫、鸡、两栖动物和爬行动物，尽管哺乳动物比如非人类的灵长类动物、啮齿类动物是优选的。
术语“治疗有效量”是指足以预防或改善与疾病或病症(例如癌症)有关的症状和/或减轻疾病或病症严重程度的本发明的抗GPC3双特异性抗体或其抗原结合片段的量。治疗有效量应在所治疗的病症的背景下理解，其中本领域技术人员可以容易地识别出实际的有效量。
本发明的抗体是如下文和以下实施例所述的在结构上和化学上进行表征的单克隆抗体。抗体的重链/轻链可变区的氨基酸序列ID号汇总在表1中，一些抗体具有相同的VH或VL，例如，hYP7和其突变体hYP7HM具有相同的轻链(hYP7-L)，h1B12和其突变体h1B12-G34R、h1B12-G34K、h1B12-G34A具有相同的重链(h1B12-H)。抗体的重链恒定区可以是具有例如以SEQ ID NO：54所示的氨基酸序列的人IgG1重链恒定区，轻链恒定区可以是具有例如以SEQ ID NO：55所示的氨基酸序列的人免疫球蛋白κ轻链恒定区。
表1中的重链可变区CDR和轻链可变区CDR由Kabat、Chothia、IMGT、AbM或Contact编号系统/方法定义。然而，如本领域所公知的，CDR区也可以基于重链/轻链可变区序列的其它诸如不同编号系统所定义的本发明抗体的CDR区详见表2。
表1 抗GPC3双特异性抗体重链/轻链可变区的氨基酸序列






表2抗GPC3双特异性抗体的重链可变区和轻链可变区CDR的编号系统/方法定义






通过下面的详尽描述和实施例，本发明所公开的其它特征和优点将变得显而易见，其不应被解释为限制性的。在本申请全文中引用的所有参考文献、Genbank条目、专利和公开的专利申请的内容以引用的方式明确地并入本文。
附图说明
1)图1双特异性抗体(重链)构建示意图。h1B12抗体的重链(1B12-VH)通过2个GGGGS接头，与第二抗体(如hYP7、h2D8)的不同scFV形式相连。其中第二抗体的VH与VL可变区之间接头可以为3个/4个GGGGS，YP7-HM指代含A68G突变的YP7-VH，IgG1-CH指代人IgG1的重链恒定区。
2)图2显示了双特异性抗体及人源化单抗与人GPC3蛋白的ELISA结合曲线，各抗体与人GPC3均具有高亲和力(EC50均在sub-nM水平)。
3)图3A和图3B显示了双特异性抗体及人源化单抗与人肝癌细胞HepG2或Hep3B的结合情况。相比bsDH系列双抗(基于h1B12+h2D8)或者各单抗，bs-FH系列双抗(基于h1B12+hYP7)具有更高的细胞亲和力(对HepG2细胞和Hep3B的EC50值均在2nM左右)。
4)图4A和图4B显示了双特异性抗体及人源化单抗在人肝癌细胞HepG2或Hep3B中的内吞情况。相比bsDH系列双抗(基于h1B12+h2D8)或者各单抗，bs-FH系列双抗(基于h1B12+hYP7)表现出更好的抗体内吞。
5)图5A和图5B显示了抗体-药物偶联物与人肝癌细胞Hep3B或Huh-7的结合情况。双特异性抗体-药物偶联物相较单抗-药物偶联物，在GPC3抗原高表达细胞株(Hep3B)和低表达细胞株(Huh-7)中均表现出更高的细胞亲和力。
6)图6A-图6D显示了抗体-药物偶联物对人肝癌细胞HepG2、Hep3B或Huh-7的杀伤效果。在高浓度(图6A-图6C，66.7nM)及低浓度下(图6D，1nM)，双特异性抗体-偶联药物相比单抗-偶联药物，表现出对肿瘤细胞的更好杀伤效果。
7)图7A和图7B显示了未偶联药物的双特异性抗体及单抗，对人肝癌细胞HepG2(图7A)或Hep3B(图7B)无直接杀伤效果。
8)图8A显示了1B12及其变体的人源化单抗与人GPC3蛋白的ELISA结合曲线。
9)图8B、8C和8D显示了1B12及其变体的人源化单抗与人肝癌细胞HepG2、Hep3B或Huh-7的结合情况。
10)图9显示了1B12人源化单抗在人肝癌细胞Hep3B中的内吞情况。
具体实施例
下面通过实施例和附图解释本发明，但本发明不局限于此。
实施例1
使用杂交瘤技术制备小鼠抗GPC3单克隆抗体
1.1免疫小鼠
根据文献E.Harlow，D.Lane，Antibodies：a laboratory manual，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY，1988.中方法对小鼠进行免疫。将人GPC3蛋白细胞外全长结构域第25位氨基酸残基Gln到559位氨 基酸残基His作为免疫原(Antigen 1)，免疫BALB/c小鼠，在第二次和第三次免疫后测试血清效价，将显示出最高抗体滴度的2只小鼠进行电融合。使用Antigen 1免疫原，或细胞外C端结构域第359位氨基酸残基Ser到559位氨基酸残基His多肽抗原(Antigen 7)，用于确定抗血清滴度和筛选分泌抗原特异性抗体的杂交瘤细胞。
1.2杂交瘤细胞融合与筛选
在细胞融合前，将鼠骨髓瘤细胞株的细胞(SP2/0-Ag14，ATCC#CRL-1581)培养至对数生长期。根据Kohler G和Milstein C，“Continuous cultures of fused cells secreting antibody of predefined specificity”，Nature，256：495-497(1975)中所述的方法，处死免疫后的小鼠，在无菌条件下取出免疫小鼠的脾脏，制备成脾细胞悬液，使用PEG化学法融合脾B细胞和处于对数生长期的鼠骨髓瘤细胞，并对融合细胞进行连续培养。随后将融合后的杂交瘤细胞铺板至96孔板中，并用含有20％FCS/HAT的DMEM培养基进行培养。通常7至10天后显微镜下可观察到存活的杂交瘤克隆。细胞铺板两周后，收集各孔杂交瘤培养上清，使用ELISA方法通过重组人GPC3蛋白(Antigen 1和Antigen 7)来筛选表达抗GPC3抗体的杂交瘤。具体筛选的操作步骤为：重组人GPC3蛋白(Antigen 1或Antigen 7，2μg/mL)包被于96孔板中，50μL/孔，4℃孵育过夜。PBST洗板3次后，每孔加入200μL封闭液(PBS+1％BSA)于37℃封闭1小时。用封闭液稀释各抗体至2μg/mL，然后3倍梯度稀释，共计八个浓度，按50μL/孔加至酶标板中，37℃孵育1小时。PBST洗板三次，然后加入预稀释的Peroxidase-conjugated affinipure F(ab’)2fragment goat anti-mouse IgG(H+L)(Jackson，参照说明书用封闭液稀释5000倍)，50μL/孔，37℃孵育1小时。PBST洗板3次，加入1-StepTM Ultra TMB-ELISA底物显色液(Thermo)，50μL/孔，室温避光显色3-5分钟。用2M HCl终止反应，50μL/孔。酶标仪450nm测定OD值。计算EC50值，筛选得到能够分泌具有高特异性结合活性抗体的杂交瘤克隆。
表3杂交瘤抗体与人GPC3蛋白结合的EC50
根据表3的结果可以看出，各小鼠单抗与GPC3蛋白均有高亲和力，大部分识别GPC3蛋白C端结构域Antigen7，而m2G12识别C端之外的结构域。
进一步的，使用ELISA方法检测了各抗体对不同种属GPC3蛋白的识别情况。Cynomolgus GPC3(ACRO Biosystems)、Mouse GPC3(ACRO Biosystems)分别包被于96孔板，浓度2μg/mL，50μL/孔，按照上述ELASA实施方法进行检测，结果显示在表4中。
表4杂交瘤抗体对猴、鼠GPC3蛋白的识别情况
备注：√代表亲和识别，×代表不识别。
进一步的，通过竞争性ELISA，确定各候选抗体是否与GC33抗体识别表位相同。具体步骤为：将各抗体稀释为2μg/mL，加入96孔酶标板中，100μL/孔，4℃包被过夜。用PBST洗板三次，加入封闭液(PBS+1％BSA)，200μL/孔，37℃封闭1h。每孔加入2μg/mL Insert 1抗原，100μL/孔，37℃孵育1h，用PBST洗板三次。用封闭液稀释GC33抗体至2μg/mL，然后3倍梯度稀释，共计八个浓度，之后把稀释好的GC33加至酶标板中，100μL/孔，37℃孵育1h。弃去上清，用PBST洗板三次，加入稀释好的Peroxidase-conjugated goat anti-human IgG(Jackson，参照说明书用封闭液稀释5000倍)，100μL/孔，37℃孵育1h。弃上清，用PBST洗板三次，加底物1-step Ultra TMB-ELISA显色液(Thermo)，50μL/孔，避光反应约4min30s后，加入50μL终止液(2M HCl)。酶标仪450nm测定OD值。分析结果汇总于表5，其中m1B12、m1H4与GC33具有相同或Overlap的抗原识别位点。
表5识别GPC3不同表位的抗体
1.3杂交瘤抗体的测序鉴定
通过5’RLM-RACE钓取杂交瘤抗体可变区基因。简言之，提取各候选杂交瘤总RNA，使用FirstChoice RLM-RACE Kit(Thermo)进行样品反转录获取cDNA，PCR扩增重链和轻链可变区基因。PCR产物克隆至载体进行测序，获得m1B12、m2D8、m2G12、m1B8、m1H4、m36C12、m37F4各抗体序列，见表6。
表6抗GPC3杂交瘤抗体可变区序列
实施例2
小鼠抗GPC3单克隆抗体的人源化改造
选择小鼠抗GPC3各单克隆抗体用于人源化和进一步的研究。如下所述，使用成熟的CDR移植方法对小鼠单克隆抗体进行人源化改造。将小鼠各单克隆抗体的轻链可变区和重链可变区序列与人类免疫球蛋白基因数据库进行BLAST比对，以选择用于各小鼠抗体人源化改造的受体框架区。选择与各小鼠抗体同源性最高的人种系IGVH和IGVK作为用于人源化改造的受体框架。将各小鼠抗体重链可变区或轻链可变区的CDR移植到所选择的人种系框架区中，并且将框架区中的氨基酸残基进行回复突变以获得更多候选的人源化抗体的重链可变区和/或轻链可变区。人源化后各抗体的重链可变区和轻链可变区，及突变位点设计汇总于表7中。
表7抗GPC3人源化抗体可变区序列

实施例3
人源化单抗及双特异性抗体的制备
3.1载体构建
首先将编码人IgG1重链恒定区(IgG1-CH)的核苷酸序列，前端接上分泌信号肽(SP)编码序列，后端接上终止密码子TAG，进行基因合成并克隆到pUC57-GW-kan载体(苏州金唯智生物科技有限公司)，通过In-fusion cloning方法插入到pCDNA3.1载体CMV启动子下游。具体地，通过设定特定插入位点引物、及高保真PCR酶(HiFi PCR Premix，TAKARA)分别扩增合成的基因片段、及pCDNA3.1载体质粒片段，胶回收后IgG1重链基因片段和线性化载体片段进行连接(In-fusion Snap Assembly Master Mix，TAKARA)，得到重链恒定区载体pCDNA3.1-IgG1。按上述实施方法，将人免疫球蛋白κ轻链恒定区(IgG-CK)编码序列，前端加信号肽(SP)编码序列后端加终止密码子TAG，基因合成后插入到pCDNA3.1载体CMV启动子下游，得到轻链恒定区载体pCDNA3.1-CK。
对于各人源化单抗表达载体的构建，分别将各自重链可变区(VH)、轻链可变区(VL)的编码序列进行基因合成，按上述In-fusion cloning方法插入到pCDNA3.1-IgG1或pCDNA3.1-CK载体的SP和恒定区之间，得到单抗的重链表达载体和轻链表达载体。
对于双特异性抗体表达载体的构建，将第二抗体的重链可变区核苷酸序列与轻链可变区核苷酸序列，通过(GGGGS)3或(GGGGS)4接头编码序列相连接，得到scFV核苷酸序列并基因合成，然后将scFV编码序列插入到上述单抗重链表达载体的SP和VH之间，得到双特异性抗体的重链表达载体。双特异性抗体的重链载体构建 示意图如图1。
对于突变体表达载体的构建，hYP7-HM是通过基因合成的方式将hYP7-VH第68位残基编码序列由GCC替换为GGC，获得重链A68G突变(丙氨酸突变为甘氨酸)。h1B12-VL(G34X)是通过基因合成的方式将h1B12-VL第34位残基编码序列由GGC替换为CGC、AAG、GCC、GTG、ATG、CTG、TAC、CCG、TCC，获得轻链G34R、G34K、G34A、G34H、G34M、G34L、G34Y、G34P和G34S系列突变(甘氨酸突变为精氨酸、赖氨酸、丙氨酸、组氨酸、蛋氨酸、亮氨酸、酪氨酸、脯氨酸和丝氨酸)；或将h1B12-VL第33位残基编码序列由AAC替换为CGC和CCG，获得轻链N33R和N33P突变(天冬酰胺突变为精氨酸和脯氨酸)。载体构建方法如前所述。
3.2细胞转染表达
按Transfection Reagent(Mirus)说明书进行转染。如下所述，ExpiCHO-S细胞(Thermo)培养于完全培养基(High Yield Expression System，含30mL/L的Poloxamer 188 solution 10％和20mL/L的L-谷氨酰胺200mM，Mirus)，在转染前24小时进行传代培养，保证第二天细胞密度为4x106个细胞/mL，转染前稀释至2x106个细胞/mL。将轻重链质粒按1：1比例各25μg，加入到12.5mL完全培养基中混匀，再加入50μL转染试剂Transfection Reagent(Mirus)，轻柔颠倒混匀后静置4分钟，然后边摇晃边逐滴加入到稀释好的50mL细胞中，并逐滴加入1mL的CHOgro-titer Enhancer(Mirus)。转染完成后立刻放入32℃、5％CO2的培养箱，每隔一天添加5％的EfficientFeed C+AGT Supplement(Thermo)，共培养7天。
3.3抗体纯化
在摇瓶中培养7天后，收集细胞上清液，4000rpm离心20分钟后取上清，用0.22um滤膜(Milipore)过滤，通过蛋白A亲和层析纯化抗体。简言之，使用20mM PB+0.15M NaCl缓冲液以5至10倍柱体积平衡HiTrap Mabselect suRe预装柱(Cytiva)，使用AKTA Avant 150层析系统(Cytiva)，将过滤后的上清液上样，之后依次用3倍柱体积的20mM PB+0.15M NaCl缓冲液、1倍柱体积的20mM PB+1M NaCl缓冲液淋洗该纯化柱，再用20mM PB洗涤至基线平稳。最后用20mM柠檬酸(20mM柠檬酸钠调pH至3.0)洗脱抗体，收集峰形200mAu-200mAu，洗脱的抗体立即用中和缓冲液(1M Tris-HCl，pH 9.0)中和，并置于1.5mL管中，-80冻存备用。
实施例4
人源化单抗及双特异性抗体的ELISA亲和力检测
应用ELISA方法测定各抗体对人GPC3蛋白的相对结合活性。具体操作为：重组人GPC3蛋白(Antigen 1，1μg/mL)按100μL/孔包被于96孔板中，4℃孵育过夜。之后用含1％BSA的PBST(含0.05％的Tween-20)于37℃封闭2小时(200μL/孔)，PBST洗涤三次，将各人源化单抗/双特异性抗体，先用含1％BSA的PBST进行系列梯度稀释，依次加入96孔板中(100μL/孔)，双特异性抗体bsFH1-bsFH6、bsDH1和bsDH2工作浓度为：20nM、10nM、10nM往下以4为倍数依次稀释8个点；人源化单抗h1B12、h2D8、hYP7HM、、h1B12-N33P-G34P、h1B12-G34Y和GC33工作浓度为：30nM、15nM、15nM往下以4为倍数依次稀释8个点。37℃孵育1小时，PBST洗涤三次。然后按100μL/孔加入Anti-Human IgG-FC-HRP(Sigma，1/30000稀释)，37℃孵育1h，PBST洗涤三次，再加入50μLTMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm-570nm测定OD值。各双特异性抗体及人源化单抗与GPC3蛋白结合的EC50值显示在表8中，结果记载于图2和图8A中。
表8双特异性抗体与人GPC3蛋白结合的EC50
根据表8、图2和图8A的结果显示，除h1B12-N33P-G34P-VL和h1B12-G34Y单抗外，其余各人源化单抗及双特异性抗体均与GPC3蛋白具有高亲和力(sub-nM水平)。
实施例5
流式细胞术检测人源化单抗和双特异性抗体与肿瘤细胞的结合
应用流式细胞术测定各抗GPC3抗体与HepG2、Hep3B或Huh-7细胞的亲和力。具体操作为：各人源化单抗或双特异性抗体，用FACS Buffer(PBS+5％FBS)进行系列梯度稀释，依次加入96孔U形板中，具体操作如下：bsFH1-bsFH6、bsDH1、bsDH2、h1B12、h1B12-G34R、h1B12-G34K、h1B12-G34A、h1B12-G34H、h1B12-G34M、h1B12-G34L、h1B12-G34S、h1B12-N33R、h1B12-N33P-G34P、h1B12-G34Y和GC33工作浓度为：300nM、200nM、200nM往下以3为倍数依次稀释8个点；h2D8和hYP7HM工作浓度为：400nM、200nM、200nM往下以3为倍数依次稀释8个点。用胰酶消化HepG2、Hep3B细胞，1000rpm离心5min，弃上清，用含FACS Buffer重悬细胞，按照2×105个细胞/孔加入96孔板中，轻轻混匀，置于冰上孵育90min。4℃ 3500rpm离心3min后弃去上清，随后加入250μL预冷FACS Buffer重悬细胞，重复离心洗涤3次。每孔加入100μL稀释好的PE anti-human IgG FC荧光二抗(BioLegend，1μL/2×105个细胞配制)，冰上避光孵育60min。弃去上清洗涤两次，最后用200μL FACS Buffer重悬细胞。使用Attune NxT流式细胞仪(Thermo)上机检测MFI值，利用GraphPad Prism软件对数据进行处理。各双特异性抗体或人源化单抗与细胞表面GPC3抗原结合的情况如图3A、图8B(HepG2细胞)和图3B、图8C(Hep3B细胞)、图8D(Huh-7细胞)以及表9和表10所示，根据图3A-3B、图8B-8D、表9和表10的结果可以看出，除h1B12-G34Y单抗外，本发明的其余单克隆抗体或者双抗对HepG2、Hep3B或Huh-7细胞均具有很好的亲和力。
表9单克隆抗体与HepG2细胞结合的EC50
表10单克隆抗体与Hep3B细胞结合的EC50
实施例6
流式细胞术检测人源化单抗/双特异性抗体的内化效率
在HepG2、Hep3B细胞中，应用流式细胞术测定各抗GPC3抗体的内化效率。具体操作为：各人源化单抗或双特异性抗体用FACS Buffer稀释为50nM，按50μL/孔加入96孔U形板中。按照实施例5的方式进行抗体和细胞的结合。各抗体和细胞以不同时间进行孵育：冰上孵育的样品设为本底0h，其余样品分别于37℃孵育1h、2h、4h，孵育完成立即转移至冰上。每孔加入200μL FACS buffer混匀，3500rpm离心3min后弃去上清，加入含有100μL预冷的PE anti-human IgG FC荧光二抗(BioLegend，1μL/2×105个细胞配制)，冰上避光孵育60min。弃去上清洗涤两次，最后用200μL FACS Buffer重悬细胞，上机检测MFI值，利用GraphPad Prism软件对数据进行处理，双特异性抗体或人源化单抗的细胞内化效率计算公式为：(1-抗体37℃ MFI值/抗体4℃ MFI值)×100％。具体结果如图4A(HepG2细胞)和图4B(Hep3B细胞)所示，根据图4A和图4B可以看出，本发明的双抗对HepG2和Hep3B细胞具有非常好的内化效率，且本发明双抗的内化速率也更快。
实施例7
流式细胞术检测人源化单抗/双特异性抗体的内化效率
在Hep3B细胞中，应用流式细胞术测定各抗GPC3抗体的内化效率。具体操作为：各人源化单抗或双特异性抗体用FACS Buffer稀释为200nM，按50μL/孔加入96孔U形板中。按照实施例5的方式进行抗体和细胞的结合。各抗体和细胞以不同时间进行孵育：冰上孵育的样品设为本底0h，其余样品分别于37℃孵育1h、2h、4h，孵育完成立即转移至冰上。每孔加入200μL FACS buffer混匀，3500rpm离心3min后弃去上清，加入含有100μL预冷的PE anti-human IgG FC荧光二抗(BioLegend，1μL/2×105个细胞配制)，冰上避光孵育60min。弃去上清洗涤两次，最后用200μL FACS Buffer重悬细胞，上机检测MFI值，利用GraphPad Prism软件对数据进行处理，实施例2或3的双特异性抗体或人源化单抗的细胞内化效率计算公式为：(1-抗体37℃ MFI值/抗体4℃ MFI值)×100％。具体结果如图9(Hep3B细胞)所示，根据图9可以看出，本发明的单克隆抗体对Hep3B细胞具有非常好的内化效率，且本发明单抗的内化速率也明显优于GC33。
实施例8
抗体-药物偶联物的制备
将单抗或双特异性抗体原液置换为20mM PBS缓冲液(pH＝7.2)并调浓度至约5mg/mL，按50∶1体积比(抗体：EDTA)加入250mM的EDTA溶液，充分混匀。之后根据不同单抗/双特异性抗体和不同linker-payload(LP)组合情况，加入1-12倍过量摩尔比(相对于抗体)的三(2-羧乙基)膦盐酸盐(TCEP)，充分混匀后置于室温(25℃)进行还原反应3小时。向上述反应液中加入适量的DMSO，再加入6-12倍过量摩尔比(相对于抗体) 的LP药物(5mM/10mM预先溶在DMSO中)，保证反应体系中DMSO的体积占比不超过15％，充分混匀后室温下反应1.5小时。然后加入N-乙酰-L-半胱氨酸(NAC)溶液，室温下静置10min终止反应。
采用超滤脱盐，将反应液转移至10KD超滤管(Millipore)中，补加PBS缓冲液(pH 6.0)，3500g离心浓缩至所需体积，补加PBS重复离心浓缩5次。将产物经0.22μm滤膜(Millipore)过滤后，-80℃保存。
经偶联反应后，得到各种形式的ADC产物，利用体积排阻色谱法(SEC)分析ADC产物纯度，利用疏水相互作用色谱(HIC)分析药物抗体偶联比(DAR)及裸抗比例。
实施例9
流式细胞术检测抗体-药物偶联物与肿瘤细胞的结合
参照实施例5的方法，应用流式细胞术测定各抗体-药物偶联物与Hep3B、Huh-7细胞的亲和力。细胞处理、抗体-药物偶联物稀释加样、流式检测分析，均按照实施例5的方式进行操作。各抗体-药物偶联物与细胞表面GPC3抗原结合的情况如图5A(Hep3B细胞)和图5B(Huh-7细胞)所示，根据图5A和图5B的结果可以看出，本发明的单抗ADC或双抗ADC与Hep3B或Huh-7细胞均具有非常好的亲和力。
实施例10
抗体-药物偶联物对肿瘤细胞的杀伤检测
使用Cell Counting Kit-8(Dojindo)测定各抗体-药物偶联物对HepG2、Hep3B、Huh-7肝癌细胞的杀伤作用。具体操作为：HepG2、Hep3B、Huh-7细胞使用10％FBS(Gibco)+DMEM培养基(Corning)培养，当细胞汇合度达75％以上，使用胰酶(0.25％Trypsin-EDTA)消化后计数，分别以1.5×104个细胞/mL、160μL/孔(2400cells/孔)铺板至96孔板，先置于37℃、5％CO2培养箱。再使用10％FBS+DMEM培养基，将各抗体-药物偶联物分别稀释至333.5nM和5nM，按40μL/孔加入到96孔板中，使终浓度分别为66.7nM(高浓度)和1nM(低浓度)，并做重复孔，之后置于37℃、5％CO2恒温培养箱培养。分别在第1、2、3、4天使用Cell Counting Kit-8检测肿瘤细胞的活率。具体杀伤结果如图6A(HepG2细胞、高浓度66.7nM)、图6B(Hep3B细胞、高浓度66.7nM)、图6C(Huh-7细胞、高浓度66.7nM)，以及图6D(HepG2细胞、低浓度1nM)所示，根据图6A、6B、6C和6D可以看出，本发明的单抗ADC或双抗ADC在高浓度或者低浓度下对HepG2、Hep3B或Huh-7细胞均具有非常好的杀伤效果。
实施例11
单抗或双特异性抗体对肿瘤细胞的杀伤检测
使用Cell Counting Kit-8(Dojindo)测定各抗GPC3抗体对HepG2、Hep3B细胞的杀伤作用。具体操作为：使用10％FBS+DMEM培养基，将各抗GPC3抗体分别稀释至400nM、80nM、16nM、3.2nM、0.64nM、0.13nM，将HepG2、Hep3B细胞分别以1×104个细胞/mL、100μL/孔(10000cells/孔)加入96孔板。再向96孔板中加入配制好的抗GPC3抗体各100uL/孔，终浓度分别为200nM、40nM、8nM、1.6nM、0.32nM、0.06nM，并做重复孔，置于37℃5％CO2恒温培养箱培养96h。使用Cell Counting Kit-8检测肿瘤细胞的活率。具体结果如图7A(HepG2细胞)、图7B(Hep3B细胞)所示，根据图7A和图7B可以看出，单纯的单抗或双抗对HepG2或Hep3B细胞几乎没有杀伤效果。
尽管本发明已通过一个或多个实施方式来描述，但是应当理解的是，本发明不限于这些实施方式，并且本发明说明书旨在涵盖落在所附权利要求的精神和宽范围内的所有替代、修改和变动。本发明所引用的所有参考文献均通过引用整体的方式并入本发明中。

Claims (24)

  1. 一种抗GPC3单克隆抗体或其抗原结合片段，其特征在于：所述抗GPC3单克隆抗体或其抗原结合片段包含重链可变区(VH)和链可变区(VL)；
    所述重链可变区(VH)包含CDR区，所述CDR区包含与氨基酸序列SEQ ID Nos：14或SEQ ID Nos：16相同的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，所述CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义；
    所述轻链可变区(VL)包含CDR区，所述CDR区包含与氨基酸序列SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60相同的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，所述CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义。
  2. 根据权利要求1所述的抗GPC3单克隆抗体或其抗原结合片段，其特征在于，所述重链可变区(VH)的CDR区选自：
    1)SEQ ID Nos：14的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    2)SEQ ID Nos：16的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    所述轻链可变区(VL)的CDR区选自：
    1)SEQ ID Nos：15的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    2)SEQ ID Nos：17的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    3)SEQ ID Nos：44的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    4)SEQ ID Nos：45的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    5)SEQ ID Nos：46的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    6)SEQ ID Nos：56的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    7)SEQ ID Nos：57的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    8)SEQ ID Nos：58的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    9)SEQ ID Nos：59的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；或
    10)SEQ ID Nos：60的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    优选地，所述重链可变区(VH)的CDR区选自：
    1)SEQ ID Nos：14的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    优选地，所述轻链可变区(VL)的CDR区选自：
    1)SEQ ID Nos：15的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    2)SEQ ID Nos：44的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    3)SEQ ID Nos：45的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    4)SEQ ID Nos：46的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
  3. 根据权利要求1或2所述的抗GPC3单克隆抗体或其抗原结合片段，其特征在于，所述重链可变区(VH)包含与氨基酸序列SEQ ID Nos：14或SEQ ID Nos：16相同的序列；
    所述轻链可变区(AVL)包含与氨基酸序列SEQ ID Nos：15、、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45或SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60的相同的序列；
    优选地，所述重链可变区(VH)包含与氨基酸序列SEQ ID Nos：14相同的序列；
    优选地，所述轻链可变区(AVL)包含与氨基酸序列SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60的相同的序列。
  4. 根据权利要求1-3任一项所述的抗GPC3单克隆抗体或其抗原结合片段，其特征在于，所述抗GPC3单克隆抗体或其抗原结合片段还包括重链(H)和轻链(L)，所述重链(H)包含与氨基酸序列SEQ ID Nos：61或SEQ ID Nos：71相同的序列；
    所述轻链(L)包含与SEQ ID Nos：62、SEQ ID Nos：63、SEQ ID Nos：64、SEQ ID Nos：65、SEQ ID Nos：66、SEQ ID Nos：67、SEQ ID Nos：68、SEQ ID Nos：69或SEQ ID Nos：70相同的序列；
    优选地，所述重链(H)包含与氨基酸序列SEQ ID Nos：61或SEQ ID Nos：71相同的序列；
    优选地，所述轻链(L)包含与SEQ ID Nos：62、SEQ ID Nos：63、SEQ ID Nos：64或SEQ ID Nos：65相同的序列。
  5. 一种抗GPC3双特异性抗体或其抗原结合片段，其特征在于：所述双特异性抗GPC3抗体或其抗原结合片段含有第一GPC3抗原结合结构域和第二GPC3抗原结合结构域，
    所述第一GPC3抗原结合结构域包含第一重链可变区(AVH)和第一轻链可变区(AVL)；
    所述第二GPC3抗原结合结构域包含第二重链可变区(BVH)和第二轻链可变区(BVL)；
    所述第一重链可变区(AVH)的CDR区选自SEQ ID Nos：14、16、34、49或51中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
    所述第一轻链可变区(AVL)的CDR区选自SEQ ID Nos：15、17、35、44、45、46、50、52、56、57、58、59或60中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
    所述第二重链可变区(BVH)的CDR区选自SEQ ID Nos：1、3、5、6、8、12、18、20、22、24、26、28、30、32或47中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的；
    所述第二轻链可变区(BVL)的CDR区选自SEQ ID Nos：2、4、7、9、10、11、13、19、21、23、25、27、29、31、33或48中任一条的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区，其中，各CDR区是根据IMGT、Kabat、Chothia、AbM或Contact定义的方案定义的。
  6. 根据权利要求5所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述第一重链可变区(AVH)的CDR区选自：
    1)SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    2)SEQ ID Nos：16(m1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    所述第一轻链可变区(AVL)的CDR区选自：
    1)SEQ ID Nos：15(h1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    2)SEQ ID Nos：17(m1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    3)SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    4)SEQ ID Nos：45(h1B12-G34K)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    5)SEQ ID Nos：46(h1B12-G34A)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102 位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    6)SEQ ID Nos：56的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    7)SEQ ID Nos：57的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    8)SEQ ID Nos：58的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    9)SEQ ID Nos：59的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    10)SEQ ID Nos：60的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    所述第二重链可变区(BVH)的CDR区选自：
    1)SEQ ID Nos：1(YP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    2)SEQ ID Nos：3(hYP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    3)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    4)SEQ ID Nos：6(YP8)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    5)SEQ ID Nos：8(YP9)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    6)SEQ ID Nos：12(YP9.1)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    7)SEQ ID Nos：18(h2DS)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    8)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    所述第二轻链可变区(BVL)的CDR区选自：
    1)SEQ ID Nos：2(YP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    2)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    3)SEQ ID Nos：7(YP8)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    4)SEQ ID Nos：9(YP9克隆9)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    5)SEQ ID Nos：10(YP9克隆10)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    6)SEQ ID Nos：11(YP9克隆1)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    7)SEQ ID Nos：13(YP9.1)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    8)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    9)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
  7. 根据权利要求5或6任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述第一重链可变区(AVH)的CDR区选自：SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    所述第一轻链可变区(AVL)的CDR区选自：
    1)SEQ ID Nos：15(h1B12)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    2)SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    3)SEQ ID Nos：45(h1B12-G34K)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    4)SEQ ID Nos：46(h1B12-G34A)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    所述第二重链可变区(BVH)的CDR区选自：
    1)SEQ ID Nos：1(YP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    2)SEQ ID Nos：3(hYP7)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    3)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106位氨基酸残基；或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    4)SEQ ID Nos：18(h2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    5)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    所述第二轻链可变区(BVL)的CDR区选自：
    1)SEQ ID Nos：2(YP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    2)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    3)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    4)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
  8. 根据权利要求5-7任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，
    所述第一重链可变区(AVH)的CDR区选自：SEQ ID Nos：14(h1B12)的26-33位、51-58位和97-104位氨基酸残基；或31-35位、50-66位和99-104位氨基酸残基；或26-32位、52-57位和99-104位氨基酸残基；或26-35位、50-59位和99-104位氨基酸残基；或30-35位、47-59位和97-103位氨基酸残基中的任一组；
    所述第一轻链可变区(AVL)的CDR区选自：SEQ ID Nos：44(h1B12-G34R)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    所述第二重链可变区(BVH)的CDR区选自：
    1)SEQ ID Nos：5(hYP7HM)的26-33位、51-60位和99-106位氨基酸残基；或31-35位、50-68位和101-106 位氨基酸残基或26-32位、52-59位和101-106位氨基酸残基；或26-35位、50-61位和101-106位氨基酸残基；或30-35位、47-61位和99-105位氨基酸残基中的任一组；
    2)SEQ ID Nos：18(h2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    3)SEQ ID Nos：20(m2D8)的26-33位、51-58位和97-103位氨基酸残基；或31-35位、50-66位和99-103位氨基酸残基；或26-32位、52-57位和99-103位氨基酸残基；或26-35位、50-59位和99-103位氨基酸残基；或30-35位、47-59位和97-102位氨基酸残基中的任一组；
    所述第二轻链可变区(BVL)的CDR区选自：
    1)SEQ ID Nos：4(hYP7)的27-38位、56-57位和95-103位氨基酸残基；或24-40位、56-62位和95-103位氨基酸残基；或30-42位、52-61位和95-102位氨基酸残基中的任一组；
    2)SEQ ID Nos：19(h2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基或30-41位、51-60位和94-101位氨基酸残基中的任一组；
    3)SEQ ID Nos：21(m2D8)的27-37位、55-56位和94-102位氨基酸残基；或24-39位、55-61位和94-102位氨基酸残基；或30-41位、51-60位和94-101位氨基酸残基中的任一组。
  9. 根据权利要求5-8任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述第一重链可变区(AVH)的CDR区选自：h1B12或m1B12中任一条的重链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区；
    所述第一轻链可变区(AVL)的CDR区选自h1B12、h1B12-G34R、h1B12-G34K、h1B12-G34A、h1B12-G34H、h1B12-G34M、h1B12-G34L、h1B12-G34S、h1B12-N33R或m1B12中任一条的轻链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区；
    所述第二重链可变区(BVH)的CDR区选自：YP7、hYP7、hYP7HM、YP8、YP9、YP9.1、h2D8或m2D8中任一条的重链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区；
    所述第二轻链可变区(BVL)的CDR区选自：YP7、hYP7、YP8、YP9、YP9.1、h2D8或m2D8中任一条的轻链可变区的CDR区，所述CDR区包括CDR1区、CDR2区和CDR3区。
  10. 根据权利要求5-9中任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，
    所述第一重链可变区(AVH)选自SEQ ID Nos：14、16、34、49或51中任一条；
    所述第一轻链可变区(AVL)选自SEQ ID Nos：15、17、35、44、45、46、50、52、56、57、58、59或60中任一条；
    所述第二重链可变区(BVH)选自SEQ ID Nos：1、3、5、6、8、12、18、20、22、24、26、28、30、32或47中任一条；
    所述第二轻链可变区(BVL)选自SEQ ID Nos：2、4、7、9、10、11、13、19、21、23、25、27、29、31、33或48中任一条。
  11. 根据权利要求5-10任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，
    第一GPC3抗原结合结构域和第二GPC3抗原结合结构域的连接关系为如下(a1)或(a2)：
    (a1)所述第一GPC3抗原结合结构域的重链可变区和轻链可变区通过接头L1连接形成scFv，所述scFv进一步通过接头L2连接至所述第二GPC3抗原结合结构域的重链或轻链；
    (a2)所述第二GPC3抗原结合结构域的重链可变区和轻链可变区通过接头L1连接形成scFv，所述scFv进一步通过接头L2连接至所述第一GPC3抗原结合结构域的重链或轻链；
    所述接头L1和L2均独立地选自(GGGGS)n，其中，n为1、2、3、4、5或6的整数；
    优选地，所述scFv进一步通过接头L2连接至所述第一或第二GPC3抗原结合结构域的重链或轻链末端；
    更优选地，所述重链或轻链末端选自重链或轻链C端或N端。
  12. 根据权利要求5-11任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述双特异性抗GPC3抗体包含第一多肽链和第二多肽链。
    其中，所述第一多肽链(重链)的结构可选自如下(b1)-(b8)中任一：
    (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH1]-[Fcx]；
    (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH1]-[Fcx]；
    (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH1]-[Fcx]；
    (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH1]-[Fcx]；
    (b5)[BVH]-[CH1]-[Fcx]-[L2]-[AVH]-[L1]-[AVL]；
    (b6)[BVH]-[CH1]-[Fcx]-[L2]-[AVL]-[L1]-[AVH]；
    (b7)[AVH]-[CH1]-[Fcx]-[L2]-[BVH]-[L1]-[BVL]；
    (b8)[AVH]-[CH1]-[Fcx]-[L2]-[BVL]-[L1]-[BVH]；
    其中，[L1]、[L2]独立地表示接头，所述接头选自(GGGGS)n，其中，n为1、2、3、4、5或6的整数；所述CH1为抗体重链恒定区的CH1；所述[Fcx]表示抗体重链恒定区的Fc结构域，包含CH2和CH3；
    所述第二多肽链(轻链)的结构可选自如下(c1)或(c2)：
    (c1)[AVL]-[CL]；
    (c2)[BVL]-[CL]；
    所述[CL]为抗体轻链恒定区。
  13. 根据权利要求5-12任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述抗GPC3双特异性抗体的第一多肽链包含：SEQ ID Nos：36；SEQ ID Nos：37；SEQ ID Nos：38；SEQ ID Nos：39；SEQ ID Nos：40；SEQ ID Nos：41；SEQ ID Nos：42或SEQ ID Nos：43中的任一条。
  14. 根据权利要求8-13任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述双特异性抗GPC3抗体的第二多肽链由轻链可变区(VL)和轻链恒定区(CL)组成，其中VL包含：SEQ ID Nos：15、SEQ ID Nos：17、SEQ ID Nos：44、SEQ ID Nos：45、SEQ ID Nos：46、SEQ ID Nos：56、SEQ ID Nos：57、SEQ ID Nos：58、SEQ ID Nos：59或SEQ ID Nos：60中的任一条，CL包含：SEQ ID Nos：55；
    更优选地，所述VL包含：SEQ ID Nos：15；SEQ ID Nos：17；SEQ ID Nos：44；SEQ ID Nos：45或SEQ ID Nos：46中的任一条，CL包含：SEQ ID Nos：55。
  15. 根据权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段，其特征在于，所述抗GPC3单克隆抗体或其抗原结合片段或者抗GPC3双特异性抗体或其抗原结合片段是人源化抗体或其抗原结合片段。
  16. 一种药物组合物，其特征在于，其包含权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段以及药学上可接受的载体。
  17. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包含权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段。
  18. 一种多核苷酸，其特征在于，所述多核苷酸编码权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段。
  19. 一种表达载体，其特征在于，所述表达载体包含权利要求18所述的多核苷酸。
  20. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求18所述的多核苷酸或权利要求19所述的表达载体。
  21. 一种抗体-药物偶联物，其特征在于，其包含权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段以及药物或毒素。
  22. 根据权利要求21所述的抗体-药物偶联物，其特征在于，所述药物或毒素选自：SN-38、MMAE、PBD dimer、DX-8951(DXd)或DUBA中的一种或多种。
  23. 一种药物组合物，其特征在于，其包含权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段或者权利要求21或22所述的抗体-药物偶联物以及一种以上药学上可接受的载体。
  24. 权利要求1-4任一项所述的抗GPC3单克隆抗体或其抗原结合片段或者权利要求5-14任一项所述的抗GPC3双特异性抗体或其抗原结合片段或权利要求16所述的药物组合物或权利要求21或22所述的抗体-药物偶联物在制备用于治疗或预防癌症的药物中的用途，所述癌症优选为肝癌。
PCT/CN2023/122405 2022-09-30 2023-09-28 抗gpc3单克隆抗体/双特异性抗体或其抗原结合片段及其用途 WO2024067764A1 (zh)

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CN105968209A (zh) * 2011-04-19 2016-09-28 美国政府(由卫生和人类服务部的部长所代表) 对磷脂酰肌醇蛋白聚糖3特异的人单克隆抗体及其用途
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CN112175085A (zh) * 2019-06-14 2021-01-05 厦门大学 抗gpc3抗体及其用途
WO2023061505A1 (en) * 2021-10-15 2023-04-20 Concept To Medicine Biotech Co., Ltd. Anti-glypican 3 antibodies

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US20190016818A1 (en) * 2015-05-27 2019-01-17 La Jolla Biologics, Inc. Antibodies against glypican-3 and their uses in cancer diagnosis and treatment
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CN109988240A (zh) * 2017-12-29 2019-07-09 安源生物科技(上海)有限公司 抗gpc-3抗体及其用途
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