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WO2024255815A1 - 一种抗gpc3抗体或抗原结合片段及其用途 - Google Patents

一种抗gpc3抗体或抗原结合片段及其用途 Download PDF

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Publication number
WO2024255815A1
WO2024255815A1 PCT/CN2024/099094 CN2024099094W WO2024255815A1 WO 2024255815 A1 WO2024255815 A1 WO 2024255815A1 CN 2024099094 W CN2024099094 W CN 2024099094W WO 2024255815 A1 WO2024255815 A1 WO 2024255815A1
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Prior art keywords
antigen
antibody
gpc3
seq
binding fragment
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French (fr)
Inventor
牟宗春
李宁
刘小红
夏小珍
彭小伦
熊晓琳
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Salubris Chengdu Biotech Co Ltd
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Salubris Chengdu Biotech Co Ltd
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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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • 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 belongs to the field of biomedicine technology, and specifically relates to an anti-GPC3 antibody or an antigen-binding fragment thereof and uses thereof.
  • 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 attached 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).
  • 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 the small molecule drug. 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.
  • Patent CN1842540B discloses an anti-GPC3 antibody, such as GC33, which has higher ADCC and CDC activities than traditional antibodies, and its antibody epitope is located in the sequence of 544 to 553 (PKDNEISTFH) at the C-terminus of GPC3, but its binding ability to the GPC3 epitope is still weak;
  • PTDNEISTFH 544 to 553
  • Patent CN10452033B discloses a high-affinity monoclonal antibody for GPC3 and its use, such as YP7, which is an antibody prepared by immunization with a peptide consisting of 50 residues (DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS), and has a high affinity for GPC3, but its affinity for GPC3 still needs to be further improved;
  • Patent CN115850492A also discloses a monoclonal antibody and polynucleotide against GPC-3 and its preparation method and application, and claims that it solves the technical problem of low affinity between GPC3 monoclonal antibody and target antigen in the prior art. However, its affinity with GPC3 still needs to be further improved.
  • the object of the present invention is to provide an anti-GPC3 antibody or an antigen-binding fragment thereof and uses thereof, so as to solve the problems of low affinity and poor cell killing effect existing in the prior art.
  • the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the anti-GPC3 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL);
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH comprises HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 regions respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR1, CDR2 and CDR3 regions of any of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9; or respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR1, CDR2 and CDR3 regions of any of the amino acid sequences shown in SEQ ID Nos: 1-2, 6-9
  • the VL comprises LCDR1, LCDR2 and LCDR3 regions, and the LCDR1, LCDR2 and LCDR3 regions respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR1, CDR2 and CDR3 regions of the amino acid sequence SEQ ID No: 3; or respectively comprise sequences that have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the CDR1, CDR2 and CDR3 regions of the amino acid sequence SEQ ID NO: 3; the mutations may be selected from insertions, deletions and/or substitutions, and the substitutions are preferably substitutions of conservative amino acids; the CDR1, CDR2 and CDR3 regions of the amino acid sequence SEQ ID No: 3 are defined according to IMGT, Kabat, Chothia, AbM or Contact.
  • the position of the mutation is selected from one or more of position 56 (D56), position 100 (Q100) or position 102 (S102) of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9. In some embodiments, the position of the mutation includes position 56 (D56) and position 102 (S102) of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9. In some preferred embodiments, the mutation is selected from D56A, D56K, One or more of Q100R or S102R. In some preferred embodiments, the mutation is selected from Q100R, S102R, D56A, D56K+Q100R, D56A+S102R or D56K+S102R.
  • the HCDR1, HCDR2 and HCDR3 regions respectively have the same sequences as the CDR1, CDR2 and CDR3 regions of the amino acid sequence shown in any one of SEQ ID Nos: 1-2, 6-9, 23-28; and the LCDR1, LCDR2 and LCDR3 regions respectively contain the same sequences as the CDR1, CDR2 and CDR3 regions of the amino acid sequence SEQ ID No: 3.
  • the VH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID Nos: 1-2, 6-9, 23-28.
  • the VL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No: 3.
  • the VH comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 1-2, 6-9, 23-28; and/or the VL comprises a sequence identical to the amino acid sequence SEQ ID NO: 3.
  • the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and a light chain constant region (CL);
  • the heavy chain constant region (CH) can be a heavy chain constant region of human IgG1;
  • the light chain constant region (CL) can be a human ⁇ light chain constant region; specifically, the heavy chain constant region (CH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence SEQ ID No: 4, 19 or 20;
  • the light chain constant region (CL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence SEQ ID No: 5.
  • the heavy chain constant region (CH) comprises a sequence identical to the amino acid sequence SEQ ID Nos: 4, 19 or 20;
  • the light chain constant region (CL) comprises a sequence identical to the amino acid sequence SEQ ID No: 5.
  • the heavy chain (H) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID Nos: 11-18, 29-34; and/or the light chain (L) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 10.
  • the heavy chain (H) comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 11-18, 29-34;
  • the light chain (L) comprises a sequence identical to the amino acid sequence SEQ ID No: 10.
  • the anti-GPC3 antibody or antigen-binding fragment thereof described herein is a humanized monoclonal antibody.
  • the anti-GPC3 antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention has one or more of the following biological functions:
  • KD equilibrium dissociation constant
  • the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to the antigen shown in SEQ ID Nos: 37 or 38, and does not bind to the antigen shown in SEQ ID No: 36.
  • the antibody or antigen-binding fragment thereof comprises: VL shown in SEQ ID No: 39 and VH shown in SEQ ID No: 40; VL shown in SEQ ID No: 41 and VH shown in SEQ ID No: 42; VL shown in SEQ ID No: 43 and VH shown in SEQ ID No: 44; VL shown in SEQ ID No: 45 and VH shown in SEQ ID No: 46; VL shown in SEQ ID No: 47 and VH shown in SEQ ID No: 48.
  • the present invention provides an anti-GPC3 antibody or an antigen-binding fragment thereof, which competes with a reference antibody for binding to the same epitope of the GPC3 protein, wherein the reference antibody comprises a heavy chain shown in SEQ ID NO: 11 and a light chain shown in SEQ ID NO: 10.
  • the anti-GPC3 antibody or antigen-binding fragment thereof is capable of blocking at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the binding of the reference antibody to the GPC3 protein.
  • the competitive binding can be determined by a competitive binding assay.
  • Competitive binding assays are well known to those skilled in the art, and are immunological assays for detecting and quantifying unknowns by the ability of unknowns to inhibit the binding of labeled known antigens and their specific antibodies, also known as competitive inhibition assays.
  • the antigen is pre-coated on a microplate, and then a serial dilution of the unlabeled test antibody and a specific concentration of the labeled known antibody are added to the pre-coated microplate for incubation, and then after washing, the amount of the known antibody bound to the plate is determined at different dilutions of the test antibody.
  • the ability of the test antibody to block the labeled reference antibody can be determined by radioimmunoassay, enzyme immunoassay such as ELISA, or fluorescent immunoassay.
  • Another object of the present invention is to provide a novel GPC3 antigen epitope peptide to solve the problem of unmet development needs of anti-GPC3 antibodies, vaccines and related diagnostic reagents in the prior art, and to provide an effective tool for developing anti-GPC3 antibodies with strong binding ability to GPC3 or good cell killing effect.
  • the present invention provides a GPC3 antigen epitope peptide, wherein the GPC3 antigen epitope peptide is immunogenic and can induce an immune response in an organism to produce antibodies against GPC3.
  • the present invention provides a GPC3 antigen epitope peptide, wherein the GPC3 antigen epitope peptide consists of at least 7 consecutive amino acid residues within residues 485-496 of a human GPC3 protein, wherein the amino acid sequence of the human GPC3 protein is shown in SEQ ID No: 35, and the GPC3 antigen epitope peptide Peptides have one or more of the following biological functions:
  • an immune response e.g., a humoral immune response
  • the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody is determined by an ELISA method.
  • the ELISA method is described in Example 12, including coating the GPC3 antigen epitope peptide on an ELISA plate, serially diluting the anti-GPC3 antibody, adding the ELISA plate to the ELISA plate for incubation, washing and coloring after the incubation is completed, and measuring the OD value at 450nm after termination.
  • the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody is reflected by the OD 450 value of the antigen-antibody complex determined by ELISA.
  • the OD 450 value of the anti-GPC3 antibody at saturation is not less than 1.5 ⁇ 0.1, or not less than 2 ⁇ 0.1, or not less than 2.5 ⁇ 0.1, or not less than 3 ⁇ 0.1. In some embodiments, the OD 450 value is not less than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9.
  • the anti-GPC3 antibody that specifically binds to the GPC3 antigen epitope peptide comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID No: 11, and the light chain comprising the amino acid sequence shown in SEQ ID No: 10.
  • the GPC3 antigen epitope peptide is 7, 8, 9, 10, 11 or 12 amino acids in length. In some embodiments, the GPC3 antigen epitope peptide comprises at least one of asparagine at position 487 and phenylalanine at position 493.
  • the GPC3 antigen epitope peptide consists of consecutive amino acid residues at residues 487-493 of the human GPC3 protein. In some preferred embodiments, the GPC3 antigen epitope peptide consists of the amino acid sequence shown in SEQ ID No: 38.
  • the present invention provides a recombinant antigen, which comprises the GPC3 antigen epitope peptide of the present invention and a carrier protein.
  • the recombinant antigen can enhance the immunogenicity of the epitope peptide, so that it can be recognized by the body's immune system and induce an immune response.
  • the recombinant antigen has one or more of the following biological functions:
  • an immune response e.g., a humoral immune response
  • the anti-GPC3 antibody that specifically binds to the recombinant antigen comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence shown in SEQ ID No: 11, and the light chain comprises the amino acid sequence shown in SEQ ID No: 10.
  • the GPC3 antigen epitope peptide of the present invention is directly connected to a carrier protein or connected via a linker.
  • the linker can be a rigid or flexible linker, such as a peptide linker, wherein the peptide linker comprises one or more serines and/or glycines.
  • the GPC3 antigen epitope peptide of the present invention is linked to the N-terminus and/or C-terminus of the carrier protein, and/or is inserted into the interior of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope peptide of the present invention is linked to the C-terminus of the carrier protein. In some preferred embodiments, the GPC3 antigen epitope of the present invention is linked to the N-terminus of the carrier protein.
  • the carrier protein includes, but is not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), thyroglobulin, fibrinogen, gelatin, multimeric antigenic peptides, including diphtheria toxin DT, diphtheria toxin transmembrane domain DTT, rotavirus VP7, Leishmania heat shock protein, Campylobacter jejuni flagellin, Chlamydia trachomatis major outer membrane protein, chicken ovalbumin (OVA) or immunoglobulin Fc domain, such as IgG1, IgG2, IgG3 or IgG4 Fc domain.
  • the carrier protein is selected from KLH and BSA.
  • the present invention provides a chimeric antigen receptor comprising the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention.
  • CAR chimeric antigen receptor
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-GPC3 antibody or antigen-binding fragment thereof, or an antibody conjugate (e.g., an antibody-drug conjugate), or an oncolytic virus, or a chimeric antigen receptor, or a bispecific or multispecific antibody molecule, or a GPC3 antigen epitope peptide, or a recombinant antigen, and one or more pharmaceutically acceptable carriers.
  • the composition comprises more than one antibody (or its antigen-binding fragment, or antibody conjugate, or oncolytic virus)
  • the antibody or its antigen-binding fragment, or antibody conjugate, or oncolytic virus
  • the composition may optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.
  • the pharmaceutical composition of the present invention may be a vaccine, including but not limited to a protein vaccine or a nucleic acid vaccine.
  • 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 are 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 kit comprising the anti-GPC3 antibody or antigen-binding fragment thereof, GPC3 antigen epitope peptide, recombinant antigen, antibody-drug conjugate, or bispecific or multispecific antibody molecule of the present invention.
  • the kit comprises the GPC3 antigen epitope peptide of the present invention, and a means for detecting antibodies.
  • the kit is used to detect anti-GPC3 antibodies. In some embodiments, the kit is used to detect whether anti-GPC3 antibodies are present in a sample. In some embodiments, the kit is used to detect the level of anti-GPC3 antibodies in a sample. In some embodiments, the anti-GPC3 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 10.
  • the present invention provides a polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof, antigenic epitope peptide or recombinant antigen of the present invention.
  • the polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences.
  • the polynucleotide is a cDNA molecule.
  • the polynucleotide of the present invention can be prepared or obtained by known means based on the information of the amino acid sequence of the present invention, for example, by automatic DNA synthesis and/or recombinant DNA technology.
  • nucleic acids encoding protein sequences include nucleic acids with codon degeneracy.
  • the amino acid sequence of the present invention can be encoded by a variety of nucleic acids.
  • the genetic code is universal and well known. Nucleic acids encoding any amino acid sequence of the present invention can be easily conceived based on the common knowledge in the art, and can be optimized for production. Although the possible number of nucleic acid sequences encoding a given amino acid is very large, given the standard table of the genetic code, and with the assistance of a calculator, those skilled in the art can easily produce every possible combination of nucleic acid sequences encoding a given amino acid.
  • the present invention provides an expression vector comprising the polynucleotide of the present invention, wherein 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 provides a host cell comprising the polynucleotide of the present invention or the aforementioned expression vector; the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, a NS0 cell or other mammalian cell, preferably a CHO cell.
  • the present invention provides a bispecific or multispecific antibody molecule comprising the anti-GPC3 antibody or antigen-binding fragment thereof according to the present invention.
  • the present invention provides an antibody-drug conjugate, which comprises the anti-GPC3 antibody or its antigen-binding fragment of 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 antibody and the drug can be coupled via a linker to form an antibody-drug conjugate (ADC).
  • ADC comprises an anti-GPC3 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 at pH less than 5.5) and linkers that degrade under reducing conditions (such as disulfide linkers).
  • Non-degradable linkers typically release the drug 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 through 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 antibody or antigen-binding fragment thereof is coupled to SN-38 via a CL2A linker.
  • the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to MMAE via a mc-VC-PAB linker.
  • the anti-GPC3 antibody or its antigen-binding fragment is coupled to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.
  • 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 anti-GPC3 antibody or antigen-binding fragment thereof is coupled to DX-8951 (DXd) via a maleimide-GGFG linker.
  • the anti-GPC3 antibody or antigen-binding fragment thereof is coupled to DUBA via a Vc-seco linker.
  • the present invention also provides a use of the anti-GPC3 antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the antibody-drug conjugate of the present invention in the preparation of a medicament for treating or preventing cancer, preferably liver cancer.
  • the present invention provides a use of the GPC3 antigen epitope peptide, recombinant antigen, nucleic acid molecule, vector or host cell in any of the following:
  • the anti-GPC3 antibody comprises a heavy chain as shown in SEQ ID NO: 11 and a light chain as shown in SEQ ID NO: 10;
  • the anti-GPC3 antibody comprises a heavy chain as shown in SEQ ID NO: 11 and a light chain as shown in SEQ ID NO: 10;
  • the disease is a GPC3-positive cancer, such as liver cancer, colorectal cancer, ovarian cancer, etc.
  • the present invention provides a method for preparing an anti-GPC3 antibody or an antigen-binding fragment thereof, comprising the step of stimulating an animal immune system with the GPC3 antigen epitope peptide, recombinant antigen, nucleic acid molecule, vector or host cell of the present invention to cause the animal to produce antibodies.
  • the animal is selected from mammals such as humans, mice, rabbits, monkeys, cows, sheep or alpacas.
  • the present invention provides a method for screening anti-GPC3 antibodies or antigen-binding fragments thereof, comprising contacting the GPC3 antigen epitope peptide of the present invention with an antibody or antigen-binding fragment thereof to be analyzed, and detecting binding of the GPC3 antigen epitope peptide to the antibody or antigen-binding fragment thereof, wherein if the GPC3 antigen epitope peptide and the antibody or antigen-binding fragment thereof show binding, the antibody or antigen-binding fragment thereof is a candidate anti-GPC3 antibody or antigen-binding fragment thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof binds to the GPC3 antigen epitope peptide, and the binding is determined by an ELISA method.
  • the ELISA method is described in Example 12, including coating the GPC3 antigen epitope peptide on an ELISA plate, serially diluting the anti-GPC3 antibody or antigen-binding fragment thereof, adding the ELISA plate to the ELISA plate for incubation, washing and coloring after the incubation is completed, and measuring the OD value at 450nm after termination.
  • the specific binding of the GPC3 antigen epitope peptide to the anti-GPC3 antibody or antigen-binding fragment thereof is reflected by the OD 450 value of the antigen-antibody complex determined by ELISA.
  • the concentration of the GPC3 antigen epitope peptide is 6 ⁇ g/mL
  • the OD 450 value of the anti-GPC3 antibody or antigen-binding fragment thereof at saturation is not less than 1.5 ⁇ 0.1, or not less than 2 ⁇ 0.1, or not less than 2.5 ⁇ 0.1, or not less than 3 ⁇ 0.1.
  • the OD 450 value is not less than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9.
  • the present invention also provides a method for preparing an anti-GPC3 antibody or an antigen-binding fragment thereof of the present invention:
  • sequence of the DNA molecule of the anti-GPC3 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 (eg, 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 anti-GPC3 antibody or antigen-binding fragment thereof of the present invention can be expressed in cells, on cell membranes, or secreted outside cells.
  • the recombinant protein is 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 using 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 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 antibody or antigen-binding fragment thereof of the present invention.
  • the method for separating and purifying the anti-GPC3 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 identified 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 antibody-drug conjugate is prepared according to a method comprising the following steps:
  • interchain disulfide bonds of the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention 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.
  • 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 endocleaved 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
  • antibody as referred to herein includes complete antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains thereof.
  • Complete antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • 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 and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), separated by more conserved regions, called framework regions (FRs).
  • CDRs complementary 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 regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.
  • antigen-binding fragment 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.
  • 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 substances and/or chemicals.
  • 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.
  • 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.
  • bispecific or multispecific refers to an antibody and/or antigen binding molecule that can specifically bind to two or more different antigenic determinants.
  • a bispecific or multispecific antibody or antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific or multispecific antibody or antigen binding molecule can simultaneously bind to two or more antigenic determinants, particularly two or more antigenic determinants expressed on two or more different cells.
  • 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 antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby accurately delivering anti-tumor therapeutic agents (such as cytotoxins or cytostatics, radioactive isotopes, small molecule chemotherapeutics, etc.) to tumor target cells, accumulating and releasing intracellularly, and achieving the purpose of accurately killing tumors.
  • ADC is also considered to be the most promising anti-tumor drug because of its suitable molecular weight, high stability, strong targeting, and low toxic side effects.
  • bispecific antibodies can also be coupled to therapeutic agents.
  • the part coupled to the antibody or bispecific antibody of the present invention to form an antibody conjugate is a cytotoxin, which refers to a substance that inhibits or prevents cell function and/or causes cell destruction, and includes small molecule cytotoxins.
  • the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.
  • antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, antigen-binding fragments, naked antibodies, conjugated antibodies, humanized antibodies, or fully human antibodies.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody.
  • epitope is also referred to as an "antigenic determinant” in the art.
  • An epitope or antigenic determinant is usually composed of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • epitope peptide refers to a peptide segment on an antigen that can be used as an epitope.
  • a single epitope peptide can be specifically recognized/bound by an antibody directed against the epitope.
  • carrier protein refers to a protein that can act as a carrier of an epitope peptide, i.e., it can insert the epitope peptide at a specific position (e.g., inside the protein, at the N-terminus or C-terminus) so that the epitope peptide can be presented, thereby enabling the epitope peptide to be recognized by antibodies or the immune system.
  • conservative amino acid generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).
  • amino acids within each of the following groups are conservative amino acid residues of each other, and the replacement of the amino acid residues within the group is a replacement of conservative amino acids:
  • identity and “sequence ... identity” are interchangeable herein and are calculated in the following manner: To determine the percentage of "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • S102R indicates that the amino acid residue serine (S) at position 102 of the parent sequence is substituted with arginine (R).
  • 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 an anti-GPC3 antibody or antigen-binding fragment thereof of the present invention sufficient to prevent or ameliorate 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 one skilled in the art can readily identify the actual effective amount.
  • the antibodies of the present invention are structurally and chemically characterized monoclonal antibodies as described below and in the following examples.
  • the amino acid sequence ID numbers (SEQ ID No:) of the heavy chain/light chain variable region and constant region of the antibodies are summarized in Table 1 and Table 5.
  • the heavy chain variable region CDRs and light chain variable region CDRs in Table 1 and Table 5 are defined by the Kabat, Chothia, IMGT, AbM or Contact numbering system/method.
  • the CDR region sequences of exemplary anti-GPC3 antibodies of the present invention are detailed in Table 3.
  • FIG. 1A and FIG. 1B show the ELISA test results of the positive monoclonal antibodies screened in Example 1 at the phage level and the GPC3-B peptide.
  • Figures 2 and 3 show the affinity of GC90 and its mutant monoclonal antibodies to GPC3 protein.
  • FIG4 shows the binding of humanized monoclonal antibodies GC90 and hYP7HM to human liver cancer cells HepG2, Hep3B and Huh-7 at different concentrations.
  • Figures 5A to 5D show the binding of GC90 and its mutant mAbs to human liver cancer cells.
  • FIG6 shows the internalization of humanized mAbs GC90 and hYP7HM in human liver cancer cells HepG2 or Hep3B at a concentration of 1 nM.
  • FIG. 7 shows the killing effect of GC90 and its mutant monoclonal antibody drug conjugates on human liver cancer cells.
  • Figures 8A and 8B show the inhibition of tumors in vivo by the antibody-drug conjugates of the present invention.
  • FIG. 9 shows the binding strength results between GC90 and each overlapping peptide detected by ELISA, wherein the amino acid sequence of the underlined portion corresponds to the specific sequence of each overlapping peptide.
  • FIG. 10 shows an amino acid sequence alignment analysis between human GPC3 protein and mouse GPC3 protein.
  • FIG11 shows the distribution of antigen recognition regions of GC90 and other reported anti-GPC3 antibodies on the GPC3 protein.
  • 0.05-0.1 mL of overnight cultured monoclonal bacterial solution was transferred to a newly prepared sterile deep-well plate (with 0.5 mL of 2YT medium containing a final concentration of 0.1 mg/mL ampicillin antibiotics), cultured to an OD 600 value of about 0.6-0.8, added a certain proportion of auxiliary phages, oscillated and mixed, and then allowed to infect at 37°C for 45 minutes, and then 0.25 mL of 2YT medium (containing 0.1 mg/mL ampicillin antibiotics and kanamycin with a final concentration of 0.05 mg/mL after addition) was added and cultured overnight at 30°C 220 rpm (16-18 hours).
  • the overnight expressed bacterial solution was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant for ELISA binding detection of GPC3-B peptide and FACS binding detection of corresponding cells to obtain positive fully human antibody clones.
  • Indirect ELISA was used to evaluate the binding ability of phage-displayed antibodies in the supernatant for GPC3-B peptide.
  • the ELISA plate was coated with 100 ⁇ l/well of 4 ⁇ g/mL of GPC3-B peptide in CBS coating reagent at 4°C overnight.
  • the plate was washed with PBST (containing 0.05% Tween) and blocked with 300 ⁇ l/well of PBS containing 3% skim milk at 37°C for 1 hour.
  • the blocking solution was then discarded, and 50 ⁇ l of gradient dilutions of each phage expression supernatant and 50 ⁇ l 0.05% PBST, as well as negative control (negative control, lPl: ipilimumab) were added to each plate and incubated at room temperature for 2 hours.
  • the plate was washed three times with 0.05% PBST and incubated with 100 ⁇ l/well of goat anti-M13 phage antibody conjugated with horseradish peroxidase (Sino-Bio) at room temperature for 45 minutes.
  • the plate was washed six times with 0.05% PBST, and then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 10 minutes.
  • ELISA was further used to identify whether the positive monoclonal antibodies obtained by screening recognized GPC3-A peptide.
  • the specific operation was as follows: GPC3-A peptide and GPC3-B peptide (6 ⁇ g/mL) were coated in a 96-well plate at 100 ⁇ L/well and incubated at 4°C overnight.
  • each positive monoclonal antibody GC008, GC010, GC011, GC025, GC035, GC037, GC053, GC067, GC139, GC147, GC90, and control antibody hYP7HM were first serially diluted with PBST containing 1% BSA and added to a 96-well plate (100 ⁇ L/well) in sequence, with the working concentrations being: 30 nM, 15 nM, 15 nM, and so on, diluted in multiples of 4 for 8 points.
  • the positive monoclonal antibody screened in this example only specifically binds to the GPC3-B peptide, while the control antibody hYP7HM only recognizes the GPC3-A peptide (consistent with the patent literature), indicating that the positive monoclonal antibody screened in this example has different antigen recognition sites compared with the control anti-GPC3 antibody hYP7HM.
  • the nucleotide sequence encoding the constant region of the heavy chain of human IgG1 was connected to the secretion signal peptide (SP) coding sequence at the front end and the termination codon TAG at the rear end, and then gene synthesis was performed and cloned into the pcDNA3.1 vector (Shanghai Sangon Biotechnology Co., Ltd.), and then inserted into the downstream of the CMV promoter of the pCHOGUN 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 stop codon TAG at the rear end, and after gene synthesis, it was inserted into the downstream of the CMV promoter of the pCHOGUN vector to obtain the light chain constant region vector pCHOGUN-CK.
  • SP signal peptide
  • each human monoclonal antibody expression vector the coding sequences of the heavy chain variable region (VH) and light chain variable region (VL) of GC90 monoclonal antibody and its optimized mutants were respectively synthesized and inserted between the SP and constant region of pCHOGUN-IgG1 or pCHOGUN-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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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, dilute the cells to 2x10 6 cells/mL to ensure that the cell density is 4x10 6 cells/mL the next day.
  • 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 (1 ⁇ g/mL) was coated in a 96-well plate at 100 ⁇ L/well and incubated at 4°C overnight. Then, the plate was 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 was first serially diluted with PBST containing 1% BSA and added to a 96-well plate (100 ⁇ L/well) in sequence.
  • the working concentrations of humanized monoclonal antibodies GC90, GC90-4mu, GC90LH1, GC90LH2, GC90LH3, GC90LH4 and GC90LH5 were: 15 nM, 3.75 nM and 3.75 nM down by 4 times for 8 points. Incubate at 37°C for 1 hour, wash three times with PBST.
  • each antibody to human GPC3 protein was determined by ELISA method.
  • the specific operation was as follows: recombinant human GPC3 protein (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 was first serially diluted with PBST containing 1% BSA and added to a 96-well plate (100 ⁇ L/well).
  • each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence.
  • FACS Buffer PBS + 5% FBS
  • the working concentration of each humanized monoclonal antibody is: 100nM, 25nM, and 6.25nM, and then diluted in 4 times for 6 points.
  • HepG2, Hep3B, and Huh7 cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended with FACS Buffer at a concentration of 2 ⁇ 10 6 /mL. 50 ⁇ L was added to each well of the 96-well U-bottom plate, and then 50 ⁇ L of the above diluted antibodies was added to each well, gently mixed, and incubated on ice for 90min. After centrifugation at 4°C and 3500rpm for 3min, the supernatant was discarded, and then 250 ⁇ L of pre-cooled FACS Buffer was added to resuspend the cells, and the centrifugation and washing were repeated twice.
  • Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2 or Hep3B cells.
  • the specific operation was as follows: each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence.
  • the specific operation was as follows: GC90-4mu, GC90, GC90LH1, GC90LH2, GC90LH3, GC90LH4 and GC90LH5, the working concentration was: 100nM, 25nM, 6.25nM and then diluted in multiples of 4 for a total of 8 points.
  • HepG2 and Hep3B cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended with FACS Buffer at a concentration of 2 ⁇ 10 6 /mL, and 50 ⁇ L was added to each well of the 96-well U-bottom plate, and then 50 ⁇ L of the above diluted antibodies was added to each well, gently mixed, and incubated on ice for 90min. After centrifugation at 4°C and 3500rpm for 3min, the supernatant was discarded, and then 250 ⁇ L of pre-cooled FACS Buffer was added to resuspend the cells, and the centrifugation and washing were repeated twice.
  • each humanized monoclonal antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence.
  • the specific operation was as follows: IgG1, GC90-4mu, GC90LH6, GC90LH7, GC90LH8, GC90LH9, GC90LH10 and GC90LH11, the working concentration was: 100nM, 25nM, 6.25nM and then diluted in multiples of 4 for a total of 8-9 points.
  • HepG2 cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended with FACS Buffer at a concentration of 2 ⁇ 10 6 /mL, and 50 ⁇ L was added to each well of the 96-well U-bottom plate, and then 50 ⁇ L of the above diluted antibodies was added to each well, gently mixed, and incubated on ice for 90min. After centrifugation at 4°C and 3500rpm for 3min, the supernatant was discarded, and then 250 ⁇ L of pre-cooled FACS Buffer was added to resuspend the cells, and the centrifugation and washing were repeated twice.
  • TCEP (2-carboxyethyl) phosphine hydrochloride
  • the single antigen solution was replaced with 20mM NaAc-HAc buffer (pH5.5) and the concentration was adjusted to 5 ⁇ 1mg/mL.
  • TCEP tris(2-carboxyethyl)phosphine
  • the required amount of TCEP was added to the reaction system, mixed well, and then reduced at 37°C for 3 hours.
  • the solution was centrifuged in a 10kD ultrafiltration centrifuge tube to change the solution into 20mM NaAc/Tris, 1mM EDTA, pH7.0 buffer (3500 ⁇ g, 4 times), and the concentration was adjusted to 5 ⁇ 1mg/mL.
  • An appropriate amount of DMSO ( ⁇ 10%) was added to the above reaction solution.
  • the required amount of LP solution (5 ⁇ 10mM, pre-dissolved in DMSO) was added to the reaction system, mixed well, and then coupled at 21 ⁇ 25°C for 1 hour. Then, N-acetylcysteine (NAC) solution was added at a molar ratio of 40:1 to antibody, and the mixture was mixed and the reaction was terminated at 21-25°C for 1 hour. After the termination reaction, the solution was replaced with 20 mM His/His-HCl, pH 6.0 buffer (3500 ⁇ g, 4 times) using a 10kD ultrafiltration centrifuge tube, and adjusted to the target concentration. After filtering through a 0.22 ⁇ m sterilizing filter, it was stored at ⁇ -20°C.
  • IgG1-Dxd and GC90-4mu-Dxd random coupling products were obtained.
  • the purity of ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody coupling ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC).
  • Antibody-drug conjugates for killing tumor cells are provided.
  • HepG2, Hep3B, and Huh7 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. They were plated to 96-well plates at 1.5 ⁇ 10 4 cells/mL and 160 ⁇ L/well (2400 cells/well), respectively, and cultured overnight at 37°C and 5% CO 2 .
  • a subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line was established.
  • Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and matrix gel to adjust the cell density to 5 ⁇ 10 7 .
  • 5 ⁇ 10 6 Hep3B cells (0.1 mL/mouse) were subcutaneously inoculated on the right front back of the experimental mice, and the tumor growth was observed regularly.
  • the mice were randomly divided and given medication according to the tumor size, with 6 mice in each group, and the day of grouping was defined as day 0.
  • 1.5 mg/kg of IgG1-PBD and GC90-4mu-PBD were injected into the tail vein. Tumor volume was measured twice a week.
  • the volume calculation formula is as follows:
  • Tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the major diameter and b represents the minor diameter)
  • a subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line was established.
  • Hep3B cells in the exponential growth phase were collected and resuspended in PBS and matrix gel at a 1:1 ratio to adjust the cell density to 5 ⁇ 10 7 .
  • 5 ⁇ 10 6 Hep3B cells (0.1 mL/mouse) were subcutaneously inoculated on the right front back of the experimental mice, and the tumor growth was observed regularly.
  • the tumor grew to an average volume greater than 100 mm 3
  • the mice were randomly divided and given medication according to the tumor size, with 6 mice in each group, and the day of grouping was defined as day 0.
  • 5 or 10 mg/kg of IgG1-DUBA, hYP7HM-DUBA, and GC90-4mu-DUBA were injected into the tail vein once. Tumor volume was measured twice a week.
  • the volume calculation formula is as follows:
  • Tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the major diameter and b represents the minor diameter)
  • Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and matrix gel to adjust the cell density to 5 ⁇ 10 7 .
  • 5 ⁇ 10 6 Hep3B cells (0.1 mL/mouse) were subcutaneously inoculated on the right front back of the experimental mice, and the tumor growth was observed regularly.
  • the tumor grew to an average volume of 80-150 mm 3 (main experimental group) or 300-500 mm 3 (satellite group)
  • the mice were randomly divided into groups according to the tumor size and mouse weight for drug administration, with 6 mice in each group (1 mouse in the satellite group IgG-PBD group).
  • the second day of grouping was defined as Day 0: the satellite group was given a single tail vein injection of 1.5 mg/kg on Day 0 GC90-4mu-PBD, IgG1-PBD, satellite group IgG-PBD group 24h after administration of the maximum amount of blood to collect serum and tumor, GC90-4mu-PBD group alternately blood collection and tumor collection (15min serum collection, 7h, D1, D2, D3, D5, D7 serum collection and tumor collection); the main experimental group Day 0 single tail vein injection of IgG1-PBD (0.6mg/kg), GC90-4mu-PBD (0.1, 0.3, 0.6mg/kg), GC90-4mu-Dxd (3, 10mg/kg), the body weight and tumor size of the mice were measured twice a week.
  • the volume calculation formula is as follows:
  • Tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the major diameter and b represents the minor diameter)
  • each GPC3 protein (6 ⁇ g/mL) was coated in a 96-well plate at 100 ⁇ L/well and incubated at 4°C overnight. Then, the plate was 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 GC90 was first serially diluted with PBST containing 1% BSA and added to the 96-well plate (100 ⁇ L/well) in sequence. The working concentrations were: 30 nM, 15 nM, 15 nM, and then diluted in 4 times for 8 points.
  • the amino acid sequence of the GPC3-B peptide was divided into multiple overlapping peptides, the amino acid sequences of the overlapping peptides are shown in Table 9, overlapping peptides were synthesized and the binding properties between each peptide and GC90 were measured using ELISA to further accurately identify the antigenic epitopes recognized by GC90.
  • the specific operation was as follows: each overlapping peptide (6 ⁇ g/mL) was coated in a 96-well plate at 100 ⁇ L/well and incubated overnight at 4°C.
  • PBST containing 1% BSA containing 0.05% Tween-20
  • the antibody has strong binding activity only with GPC3-B3 or a long fragment containing GPC3-B3, and it can be determined that the GPC3 antigen epitope region is located in GPC3-B3, that is, residues 485-496 (DKNLDEEGFESG) of the human GPC3 protein.
  • FIG11 summarizes the antigen recognition regions of various anti-GPC3 antibodies on the GPC3 protein, and it can be seen that the antigen epitope bound by the antibody of the present invention is far away from the antigen binding region of other reported anti-GPC3 antibodies, and it recognizes a completely new GPC3 antigen epitope.
  • the present invention identifies a novel GPC3 antigen epitope, and the antibody that binds to the epitope has high affinity and internalization efficiency.
  • the use of the GPC3 antigen epitope to prepare immunogens has opened up a new research and development direction for the field of anti-GPC3 antibodies, which can reduce the uncertainty of antibody preparation and screening, and is conducive to the development of new antibodies with improved functions.

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Abstract

本发明提供了一种抗GPC-3抗体及其抗原结合片段，还提供了编码所述抗体的多核苷酸，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

一种抗GPC3抗体或抗原结合片段及其用途 技术领域
本发明属于生物医药技术领域，具体涉及一种抗GPC3抗体或其抗原结合片段及其用途。
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)。
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。
目前已报道的抗GPC3抗体有：
专利CN1842540B公开了一种抗GPC3抗体，例如GC33，与传统抗体相比具有较高ADCC活性和CDC活性，其抗体表位位于GPC3 C-末端的第544到第553(PKDNEISTFH)的序列内，但是对GPC3表位的结合能力仍然较弱；
专利CN10452033B公开了一种磷脂酰肌醇蛋白聚糖3的高亲和力单克隆抗体及其用途，例如YP7，其是由50个残基组成的肽免疫制备获得的抗体(DGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS)，对GPC3具有较高的亲和力，但是其与GPC3的亲和力仍有待于进一步提高；
专利CN115850492A也公开了一种抗磷脂酰肌醇蛋白多糖-3的单克隆抗体、多核苷酸及其制备方法和应用，并声称其解决了现有技术中GPC3单克隆抗体和目标抗原之间的亲和力较低的技术问题，然而其与GPC3的亲和力仍有待于进一步提高。
由此可见，现有技术报道的抗GPC3抗体仍然存在亲和力不够(尤其是细胞水平亲和力)或者杀伤力较差等问题，因此，目前，亟需开发一种对GPC3具有很强的结合能力，且具有较好的细胞杀伤效果的抗GPC3抗体或抗原结合片段。
发明内容
本发明的目的在于提供一种抗GPC3抗体或其抗原结合片段及其用途，以解决现有技术中存在的亲和力低，细胞杀伤效果差等问题。
一个方面，本发明提供一种抗GPC3抗体或其抗原结合片段，所述抗GPC3抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)；
所述VH包含HCDR1、HCDR2和HCDR3区，且所述HCDR1、HCDR2和HCDR3区分别包含与SEQ ID Nos：1-2、6-9任一所示氨基酸序列的CDR1、CDR2和CDR3区至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或分别包含与SEQ ID Nos：1-2、6-9任一所示氨基酸序列的CDR1、CDR2和CDR3区相比发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；所述氨基酸序列SEQ ID Nos：1-2、6-9的CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义；
所述VL包含LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3区分别包含与氨基酸序列SEQ ID No：3的CDR1、CDR2和CDR3区至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或分别包含与氨基酸序列SEQ ID NO：3的CDR1、CDR2和CDR3区相比发生至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；所述氨基酸序列SEQ ID No：3的CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义。
在一些实施方案中，所述突变的位置选自SEQ ID Nos：1-2、6-9任一所示氨基酸序列的第56位(D56)、第100位(Q100)或第102位(S102)中的一个或多个。在一些实施方案中，所述突变的位置包括SEQ ID Nos：1-2、6-9任一所示氨基酸序列的第56位(D56)和第102位(S102)。在一些优选实施方案中，所述突变选自D56A、D56K、 Q100R或S102R中的一个或多个。在一些优选实施方案中，所述突变选自Q100R、S102R、D56A、D56K+Q100R、D56A+S102R或D56K+S102R。
在一些实施方案中，所述HCDR1、HCDR2和HCDR3区分别具有与SEQ ID Nos：1-2、6-9、23-28任一所示氨基酸序列的CDR1、CDR2和CDR3区相同的序列；以及所述LCDR1、LCDR2和LCDR3区分别包含与氨基酸序列SEQ ID No：3的CDR1、CDR2和CDR3区相同的序列。
在一些实施方案中，所述VH包含与SEQ ID Nos：1-2、6-9、23-28任一至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。在一些实施方案中，所述VL包含与SEQ ID No：3至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些实施方式中，所述VH包含与氨基酸序列SEQ ID Nos：1-2、6-9、23-28任一相同的序列；和/或所述VL包含与氨基酸序列SEQ ID NO：3相同的序列。
在一些实施方案中，所述抗体或其抗原结合片段还包含重链恒定区(CH)和轻链恒定区(CL)；所述重链恒定区(CH)可以为人IgG1的重链恒定区；所述轻链恒定区(CL)可以为人κ轻链恒定区；具体地，所述重链恒定区(CH)包含与氨基酸序列SEQ ID Nos：4、19或20至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；所述轻链恒定区(CL)包含与氨基酸序列SEQ ID No：5至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些实施方式中，所述重链恒定区(CH)包含与氨基酸序列SEQ ID Nos：4、19或20相同的序列；所述轻链恒定区(CL)包含与氨基酸序列SEQ ID No：5相同的序列。
在一些实施方式中，所述重链(H)包含与氨基酸序列SEQ ID Nos：11-18、29-34任一至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；和/或所述轻链(L)包含与氨基酸序列SEQ ID No：10至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
作为本发明的优选技术方案，所述重链(H)包含与氨基酸序列SEQ ID Nos：11-18、29-34任一相同的序列；所述轻链(L)包含与氨基酸序列SEQ ID No：10相同的序列。
在一些实施方案中，本发明所述抗GPC3抗体或其抗原结合片段是人源化单克隆抗体。
在一些具体的实施方式中，所述抗GPC3抗体或其抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些实施方案中，本发明所述抗GPC3抗体或其抗原结合片段具有以下一种或多种生物学功能：
)与SEQ ID Nos：37或38所示的抗原特异性结合，并且不结合SEQ ID No：36所示的抗原；
)与人、猴GPC3蛋白特异性结合，并且不结合鼠GPC3蛋白。
在一些实施方案中，所述抗GPC3抗体或其抗原结合片段与所述抗原的特异性结合通过平衡解离常数KD为例如10-4M或更小(例如10-5M、10-6M、10-7M、10-8M、10-9M、10-10M、10-11M或10-12M)来表现。
在一方面，本发明提供一种抗GPC3抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性结合SEQID Nos：37或38所示的抗原，并且不结合SEQ ID No：36所示的抗原。在一些实施方案中，所述抗体或其抗原结合片段包含：SEQ ID No：39所示的VL和SEQ ID No：40所示的VH；SEQ ID No：41所示的VL和SEQ ID No：42所示的VH；SEQ ID No：43所示的VL和SEQ ID No：44所示的VH；SEQ ID No：45所示的VL和SEQID No：46所示的VH；SEQ ID No：47所示的VL和SEQ ID No：48所示的VH；SEQ ID No：49所示的VL和SEQ ID No：50所示的VH；SEQ ID No：51所示的VL和SEQ ID No：52所示的VH；SEQ ID No：53所示的VL和SEQ ID No：54所示的VH；SEQ ID No：55所示的VL和SEQ ID No：56所示的VH；或者，SEQ ID No：57所示的VL和SEQ ID No：58所示的VH。
在一方面，本发明提供一种抗GPC3抗体或其抗原结合片段，所述抗体或其抗原结合片段与参考抗体竞争结合GPC3蛋白的相同表位，所述参考抗体包含SEQ ID NO：11所示的重链以及SEQ ID NO：10所示的轻链。
在一些实施方案中，所述抗GPC3抗体或其抗原结合片段能够阻断参考抗体与GPC3蛋白的结合的至少50％、60％、70％、80％、90％、95％或99％。所述竞争结合可以通过竞争性结合试验测定。本领域技术人员熟知的竞争性结合试验，其通过未知物抑制标记的已知抗原和其特异性抗体结合的能力来检测和定量未知物的免疫学试验，也称为竞争性抑制试验。例如，将抗原预包被在微孔板上，然后把系列稀释的未标记的待测抗体以及特定浓度的经标记的已知抗体共同加入上述预包被后的微孔板中进行孵育，然后在洗涤后测定在不同稀释度的待测抗体下，已知抗体结合到板上的数量。待测抗体竞争已知抗体结合抗原的能力越强，已知抗体结合抗原的能力就越弱，结合到板上的已知抗体就越少。可以利用放射性免疫试验、酶免疫试验如ELISA、或荧光免疫试验测定待测抗体阻断经标记的参考抗体的能力。
本发明的另一目的在于提供一种新颖的GPC3抗原表位肽，以解决现有技术中存在的抗GPC3抗体、疫苗以及相关诊断试剂的开发需求未被满足的缺陷，为开发对GPC3具有强结合能力或具有较好的细胞杀伤效果的抗GPC3抗体提供有效的工具。
一个方面，本发明提供一种GPC3抗原表位肽，所述GPC3抗原表位肽具有免疫原性，能够诱导机体的免疫反应，产生针对GPC3的抗体。
一个方面，本发明提供一种GPC3抗原表位肽，所述GPC3抗原表位肽由人GPC3蛋白残基485-496内的至少7个连续氨基酸残基组成，所述人GPC3蛋白的氨基酸序列如SEQ ID No：35所示，并且所述GPC3抗原表位 肽具有如下一种或多种生物学功能：
(1)与抗GPC3抗体特异性结合；
(2)在受试者体内诱导针对GPC3的免疫应答(例如体液免疫应答)；
(3)在受试者体内诱发抗GPC3抗体的产生；
(4)预防和/或治疗受试者与GPC3相关的疾病。
在一些实施方案中，所述GPC3抗原表位肽与抗GPC3抗体的特异性结合通过ELISA方法测定。在一些实施方案中，ELISA方法参照实施例12所记载，包括将所述GPC3抗原表位肽包被于酶标板，系列梯度稀释抗GPC3抗体，加入酶标板中孵育，孵育完成后清洗、显色，终止后测定450nm的OD值。在一些实施方案中，所述GPC3抗原表位肽与抗GPC3抗体的特异性结合通过ELISA所测定的抗原抗体复合物的OD450值反映。在一些实施方案中，当所述GPC3抗原表位肽浓度为6μg/mL，抗GPC3抗体饱和时的OD450值不低于1.5±0.1，或者不低于2±0.1，或者不低于2.5±0.1，或者不低于3±0.1。在一些实施方案中，所述OD450值不低于1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8或3.9。在一些实施方案中，与所述GPC3抗原表位肽特异性结合的抗GPC3抗体包含重链和轻链，所述重链包含SEQ ID No：11所示的氨基酸序列，所述轻链包含SEQ ID No：10所示的氨基酸序列。
在一些实施方案中，所述GPC3抗原表位肽的长度为7个、8个、9个、10个、11个或12个氨基酸。在一些实施方案中，所述GPC3抗原表位肽至少包含第487位的天冬酰胺和第493位的苯丙氨酸中的一个以上。
在一些实施方案中，所述GPC3抗原表位肽由人GPC3蛋白残基487-493位的连续氨基酸残基组成。在一些优选实施方案中，所述GPC3抗原表位肽由SEQ ID No：38所示的氨基酸序列组成。
在一方面，本发明提供一种重组抗原，其包含本发明的GPC3抗原表位肽和载体蛋白。所述重组抗原能够增强所述表位肽的免疫原性，使其被机体免疫系统识别并诱导免疫应答。
在一些实施方案中，所述重组抗原具有如下一种或多种生物学功能：
(1)与抗GPC3抗体特异性结合；
(2)在受试者体内诱导针对GPC3的免疫应答(例如体液免疫应答)；
(3)在受试者体内诱发抗GPC3抗体的产生；
(4)预防和/或治疗受试者与GPC3相关的疾病。
在一些实施方案中，与所述重组抗原特异性结合的抗GPC3抗体包含重链和轻链，所述重链包含SEQ ID No：11所示的氨基酸序列，所述轻链包含SEQ ID No：10所示的氨基酸序列。
在一些实施方案中，本发明的GPC3抗原表位肽直接连接或通过接头连接载体蛋白。在一些实施方案中，所述接头可以是刚性或柔性接头，例如肽接头，所述肽接头包含一个或多个丝氨酸和/或甘氨酸。
在一些实施方案中，本发明的GPC3抗原表位肽连接至所述载体蛋白的N末端，和/或C末端，和/或插入载体蛋白的内部。在一些优选实施方案中，本发明的GPC3抗原表位肽连接至所述载体蛋白的C末端。在一些优选实施方案中，本发明的GPC3抗原表位连接至所述载体蛋白的N末端。
在一些实施方案中，所述载体蛋白包括但不限于匙孔血蓝蛋白(KLH)、牛血清白蛋(BSA)、甲状腺球蛋白、纤维蛋白原、明胶、多聚抗原肽、括白喉毒素DT、白喉毒素的跨膜结构域DTT、轮状病毒VP7、利什曼原虫的热休克蛋白、空肠弯曲菌鞭毛蛋白、沙眼衣原体主要外膜蛋白、鸡卵白蛋白(OVA)或免疫球蛋白Fc结构域，例如IgG1、IgG2、IgG3或IgG4的Fc结构域。在一些优选实施方案中，载体蛋白选自KLH和BSA。
在一方面，本发明提供一种嵌合抗原受体，其包含本发明的抗GPC3抗体或其抗原结合片段。
在另一方面，本发明提供了一种嵌合抗原受体(CAR)，其包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含本发明的抗GPC3抗体或其抗原结合片段。
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的抗GPC3抗体或其抗原结合片段，或抗体缀合物(例如抗体-药物偶联物)，或溶瘤病毒，或嵌合抗原受体，或双特异性或多特异性抗体分子，或GPC3抗原表位肽，或重组抗原，以及一种以上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、或抗体缀合物、或溶瘤病毒)时，可以分批施用抗体(或其抗原结合片段、或抗体缀合物、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。
本发明的药物组合物可以是疫苗，所述疫苗包括但不限于蛋白疫苗或核酸疫苗。
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。
在另一个方面，本发明提供一种试剂盒，其包含本发明的抗GPC3抗体或其抗原结合片段、GPC3抗原表位肽、重组抗原、抗体-药物偶联物或双特异性或多特异性抗体分子。
在一些实施方案中，所述试剂盒包含本发明的GPC3抗原表位肽，以及用于检测抗体的工具。
在一些实施方案中，所述试剂盒用于检测抗GPC3抗体。在一些实施方案中，所述试剂盒用于检测样品中是否存在抗GPC3抗体。在一些实施方案中，所述试剂盒用于检测样品中抗GPC3抗体的水平。在一些实施方案中，所述抗GPC3抗体包含重链和轻链，所述重链包含SEQ ID NO：11所示的氨基酸序列，所述轻链包含SEQ ID NO：10所示的氨基酸序列。
在另一方面，本发明提供一种多核苷酸，其编码本发明的抗GPC3抗体或其抗原结合片段、抗原表位肽或重组抗原。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。本发明的多核苷酸可基于本发明的氨基酸序列的信息通过已知的方式制备或获得，例如通过自动DNA合成和/或重组DNA技术。
如本领域中所熟知，多个密码子可编码相同氨基酸。因此，编码蛋白质序列的核酸包括具有密码子简并性的核酸。本发明所述的氨基酸序列可由多种核酸编码。遗传密码为通用且熟知。编码本发明所述的任何氨基酸序列的核酸可基于本领域的公知常识容易地构思出，且可经优化以进行生产。尽管编码给定氨基酸的核酸序列的可能数目很大，给定遗传密码的标准表下，且在计算器的辅助下，本领域技术人员可容易地产生编码给定氨基酸的核酸序列的每种可能的组合。
在另一方面，本发明提供一种表达载体，其包含本发明的多核苷酸。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。
在另一方面，本发明提供一种宿主细胞，其包含本发明的多核苷酸或前述的表达载体；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、NS0细胞或其它哺乳动物细胞，优选为CHO细胞。
在另一方面，本发明提供一种双特异性或多特异性抗体分子，其包含本发明所述的抗GPC3抗体或其抗原结合片段。
在另一方面，本发明提供一种抗体-药物偶联物，其包含本发明的抗GPC3抗体或其抗原结合片段以及药物或毒素；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。
所述抗体和所述药物可以通过接头偶联从而形成抗体-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抗体或其抗原结合片段通过CL2A接头偶联SN-38。
在本发明中，所述抗GPC3抗体或其抗原结合片段通过mc-VC-PAB接头偶联MMAE。
在本发明中，所述抗GPC3抗体或其抗原结合片段通过maleimide-dPEG8-VA-PABA接头偶联PBD dimer。
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。
在本发明中，所述抗GPC3抗体或其抗原结合片段通过maleimide-GGFG接头偶联DX-8951(DXd)。
在本发明中，所述抗GPC3抗体或其抗原结合片段通过Vc-seco接头偶联DUBA。
在另一方面，本发明还提供一种本发明的抗GPC3抗体或其抗原结合片段或本发明的药物组合物或本发明的抗体-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。
在另一方面，提供本发明的GPC3抗原表位肽、重组抗原、核酸分子、载体或宿主细胞在如下任一中的应用：
(1)制备抗GPC3抗体或其抗原结合片段；
(2)制备用于治疗和/或预防和/或诊断受试者与GPC3相关的疾病的产品；优选的，所述疾病为GPC3阳性癌症；
(3)制备检测抗GPC3抗体或其抗原结合片段的产品；优选的，所述抗GPC3抗体包含SEQ ID NO：11所示的重链和SEQ ID NO：10所示的轻链；
(4)检测抗GPC3抗体或其抗原结合片段；优选的，所述抗GPC3抗体包含SEQ ID NO：11所示的重链和SEQ ID NO：10所示的轻链；
(5)筛选抗GPC3抗体或其抗原结合片段。
在一些实施方案中，所述疾病为GPC3阳性癌症，例如肝癌、结直肠癌、卵巢癌等。
在一方面，本发明提供一种制备抗GPC3抗体或其抗原结合片段的方法，包括本发明的GPC3抗原表位肽、重组抗原、核酸分子、载体或宿主细胞刺激动物免疫系统，使所述动物产生抗体的步骤。
在一些实施方案中，所述动物选自人，鼠，兔，猴，牛，羊或羊驼等哺乳动物。
在一方面，本发明提供一种筛选抗GPC3抗体或其抗原结合片段的方法，使本发明的GPC3抗原表位肽与要分析的抗体或其抗原结合片段接触，检测所述GPC3抗原表位肽对所述抗体或其抗原结合片段的结合，其中，如果所述GPC3抗原表位肽和所述抗体或其抗原结合片段展现出结合，那么所述抗体或其抗原结合片段是候选的抗GPC3抗体或其抗原结合片段。
在一些实施方案中，所述抗GPC3抗体或其抗原结合片段与所述GPC3抗原表位肽结合，所述结合通过ELISA方法测定。在一些实施方案中，ELISA方法参照实施例12所记载，包括将所述GPC3抗原表位肽包被于酶标板，系列梯度稀释抗GPC3抗体或其抗原结合片段，加入酶标板中孵育，孵育完成后清洗、显色，终止后测定450nm的OD值。在一些实施方案中，所述GPC3抗原表位肽与抗GPC3抗体或其抗原结合片段的特异性结合通过ELISA所测定的抗原抗体复合物的OD450值反应。在一些实施方案中，当所述GPC3抗原表位肽浓度为6μg/mL，抗GPC3抗体或其抗原结合片段饱和时的OD450值不低于1.5±0.1，或者不低于2±0.1，或者不低于2.5±0.1，或者不低于3±0.1。在一些实施方案中，所述OD450值不低于1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8或3.9。进一步地，本发明还提供一种本发明的抗GPC3抗体或其抗原结合片段的制备方法：
本发明所述的抗GPC3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。
本发明的抗GPC3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抗体或其抗原结合片段。
作为本发明所述抗GPC3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。
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：
本发明的抗GPC3抗体或其抗原结合片段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。
为清楚起见，本文定义了在化合物的描述中所使用的通用术语。
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
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。
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“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。
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。
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。
术语“双特异性”或“多特异性”指抗体和/或抗原结合分子能够特异性结合两种或多种不同的抗原性决定簇，通常，双特异性或多特异性抗体或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性或多特异性抗体或抗原结合分子能够同时结合两种或多种抗原决定簇，特别是在两种或多种不同的细胞上表达的两种或多种抗原性决定簇。
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。
如本文所用，术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
本文术语“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、抗原结合片段、裸抗体、缀合抗体、人源化抗体或全人抗体。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，EpitopeMapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“表位肽”是指，抗原上能够用作表位的肽段。在一些情况下，单独的表位肽即能够被针对所述表位的抗体特异性识别/结合。在另一些情况下，可能需要将表位肽与载体蛋白融合，以便表位肽能够被特异性抗体识别。
如本文中所使用的，术语“载体蛋白”是指这样的蛋白，其可以充当表位肽的载体，即，其可以在特定位置处(例如蛋白内部，N端或C端)插入表位肽，以便该表位肽能够呈现出来，从而该表位肽能够被抗体或免疫系统识别。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”和“序列……一致性”可以互换，通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
如本文所用，符号“+”表示突变的组合。在本文中，使用以下术语指定突变：S102R表示亲本序列第102位的氨基酸残基丝氨酸(S)被精氨酸(R)取代。
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/轻链可变区及恒定区的氨基酸序列ID号(SEQ ID No：)汇总在表1、表5中。
表1、表5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。本发明示例性的抗GPC3抗体的CDR区序列详见表3。
表1抗GPC3抗体重链/轻链可变区及恒定区的氨基酸序列



表2 GPC3抗原的氨基酸序列

表3抗GPC3抗体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
图1A至图1B显示了实施例1筛选的阳性单克隆抗体在噬菌体水平与GPC3-B肽的ELISA检测结果。
图2、图3显示了GC90及其突变体单抗对GPC3蛋白的亲和力。
图4显示了人源化单抗GC90和hYP7HM在不同浓度下与人肝癌细胞HepG2、Hep3B和Huh-7的结合情况。
图5A至5D显示了GC90及其突变体单抗与人肝癌细胞的结合情况。
图6显示了人源化单抗GC90和hYP7HM在1nM浓度下在人肝癌细胞HepG2或Hep3B中的内吞情况。
图7显示了GC90及其突变体单抗的药物偶联物对人肝癌细胞的杀伤情况。
图8A、8B显示了本发明的抗体-药物偶联物对体内肿瘤的抑制情况。
图9显示了GC90与各重叠肽之间经ELISA检测所反应的结合强度结果，其中下划线部分的氨基酸序列对应各重叠肽的具体序列。
图10显示了人GPC3蛋白和鼠GPC3蛋白之间的氨基酸序列比对分析。
图11显示了GC90与其他已报道的抗GPC3抗体在GPC3蛋白上的抗原识别区域分布情况。具体实施例
实施例1
靶向GPC3新表位的全人源抗体阳性克隆分子获得
使用天然全人噬菌体展示文库与GPC3-B肽(GPC3蛋白细胞外结构域第478位氨基酸残基Met到531位氨基酸残基Asp，作为抗原对人源化重组抗体库进行筛选)进行三轮淘选，获得噬菌体文库洗脱液。将中和后的噬菌体淘选洗脱液加入准备好的TG1菌液中，混匀后37℃静置侵染TG1宿主菌45分钟。充分侵染后将菌液梯度稀释涂布在带有相关抗性的琼脂平板上，倒置于37℃过夜培养。次日，准备分装有0.5mL 2YT培养基(含有0.2％w/v的葡萄糖，及0.1mg/mL氨苄抗生素)的96孔无菌深孔板，用无菌枪头挑取平板中培养的单克隆菌落于对应孔板中，37℃220rpm振荡过夜培养(16-18小时)。次日取0.05-0.1mL过夜培养的单克隆菌液转接于新准备的无菌深孔板中(分装有0.5mL含有终浓度0.1mg/mL氨苄抗生素的2YT培养基)，培养至OD600值约为0.6-0.8，加入一定比例的辅助噬菌体振荡混匀后于37℃静置侵染45分钟后，补加0.25mL 2YT培养基(含有0.1mg/mL氨苄抗生素，及加入后终浓度为0.05mg/mL的卡那霉素)并于30℃220rpm振荡过夜培养(16-18小时)。将过夜表达的菌液4000rpm离心10分钟，获得噬菌体展示表达上清用于针对GPC3-B肽的ELISA结合检测及针对相应细胞的FACS结合检测，以获得阳性全人源抗体克隆。
间接ELISA用于评估上清液中噬菌体展示抗体对于GPC3-B肽的结合能力。将ELISA酶标板用100μl/孔的CBS包被试剂中4μg/mL的GPC3-B肽在4℃下包被过夜。用PBST(含0.05％吐温)洗涤板，并将其用300μl/孔的含3％脱脂牛奶的PBS在37℃封闭1小时。随后弃去封闭液，向每个板加入50μl梯度稀释的各噬菌体表达上清液及50μl 0.05％ PBST，以及阴性对照(negative control，lPl：伊匹单抗)，然后在室温下孵育2小时。将板用0.05％ PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗M13噬菌体抗体(义翘)室温孵育45分钟。将板用0.05％ PBST洗涤六次，然后加入TMB显色液(金斯瑞)于室温避光孵育10分钟。通过加入50μl的1M HCl终止液(西格玛)终止反应。使用酶标仪在450nm下读板。结合能力检测结果如图1A-1B所示，显示了多个阳性单克隆抗体在噬菌体水平与GPC3-B肽具有良好的结合能力。
进一步采用ELISA鉴定了筛选获得的阳性单克隆抗体是否识别GPC3-A肽，具体操作如下：将GPC3-A肽和GPC3-B肽(6μg/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，将各阳性单克隆抗体GC008、GC010、GC011、GC025、GC035、GC037、GC053、GC067、GC139、GC147、GC90，以及对照抗体hYP7HM，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)，其工作浓度为：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50μL TMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm测定OD值。结果如表4所示，本实施例筛选出的阳性单克隆抗体仅特异性地结合GPC3-B肽，而对照抗体hYP7HM仅识别GPC3-A肽(与专利文献记载一致)，表明本实施例筛选出的阳性单克隆抗体与对照抗GPC3抗体hYP7HM相比具有不同的抗原识别位点。
表4抗GPC3抗体与GPC3-A肽、GPC3-B肽的结合情况
注：“-”指示无结合；“++++”指示ELISA检测OD450值大于1.5。
本实施例筛选的阳性克隆进行测序，抗体的重链可变区和轻链可变区氨基酸序列见表5。
表5抗GPC3抗体的轻链和重链可变区序列

实施例2
人源化单抗的制备
2.1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pcDNA3.1载体(生工生物工程(上海)股份有限公司)，通过In-fusion cloning方法插入到pCHOGUN载体CMV启动子下游。具体地，通过设计特定插入位点引物、及高保真PCR酶 (HiFi PCR Premix，TAKARA)分别扩增合成的基因片段、及pCHOGUN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pCHOGUN-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pCHOGUN载体CMV启动子下游，得到轻链恒定区载体pCHOGUN-CK。
对于各人源单抗表达载体的构建，分别将GC90单抗及其优化突变体各自的重链可变区(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pCHOGUN-IgG1或pCHOGUN-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。具体各抗体序列，见表6。
表6人源化单抗的轻链和重链序列



2.2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 188solution 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，将细胞稀释至2x106个细胞/mL，以确保第二天细胞密度为4x106个细胞/mL。将轻重链质粒按1：1比例各25μg，加入至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Efficient Feed C+AGT Supplement(Thermo)，共培养7天。
2.3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3
人源化单抗的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(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，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)，人源化单抗GC90、GC90-4mu、GC90LH1、GC90LH2、GC90LH3、GC90LH4和GC90LH5工作浓度为：15nM、3.75nM以及3.75nM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50μL TMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm-570nm测定OD值。根据图2的结果可以看出，本发明的GC90单抗及其突变体与GPC3蛋白均具有非常好的结合力。
实施例4
亲和成熟单抗分子ELISA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(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，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)，人源化单抗GC90-4mu、GC90LH6、GC90LH7、GC90LH8工作浓度为：10000ng/ul、3333.33ng/ul以及1111.11ng/ul往下依次3倍稀释共10个点，以及GC90LH8和GC90LH10工作浓度为：10000ng/ul、2500ng/ul以及625ng/ul往下依次4倍稀释共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50μL TMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm-570nm测定OD值。根据图3的结果可以看出，本发明的GC90单抗及其突变体与GPC3蛋白均具有非常好的结合力。
实施例5
流式细胞术检测人源化单抗与肿瘤细胞的结合
应用流式细胞术测定各抗GPC3抗体与HepG2、Hep3B、Huh7细胞的亲和力。具体操作为：各人源化单抗，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：各人源化单抗的工作浓度为：100nM、25nM以及6.25nM往下以4为倍数依次稀释6个点。用胰酶消化HepG2、Hep3B、Huh7细胞，1000rpm离心5min，弃上清，用含FACS Buffer重悬细胞，浓度为2×106个/mL，96孔U底板中每孔加入50μL，再将上述稀释好的抗体每孔加入50μL，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2次。每孔加入100μL稀释好的PE anti-human IgG FC荧光二抗(BioLegend，0.5μL/1×105个细胞配制)，冰上避光孵育30min。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，图4结果表明本发明的单抗对HepG2细胞、Hep3B细胞和Huh-7细胞均具有很好的亲和力。
实施例6
流式细胞术检测人源化单抗与肿瘤细胞的结合
应用流式细胞术测定各抗GPC3抗体与HepG2或Hep3B细胞的亲和力。具体操作为：各人源化单抗，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：GC90-4mu、GC90、GC90LH1、GC90LH2、GC90LH3、GC90LH4和GC90LH5，工作浓度为：100nM、25nM、6.25nM往下以4为倍数依次稀释共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，浓度为2×106个/mL，96孔U底板中每孔加入50μL，再将上述稀释好的抗体每孔加入50μL，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2次。每孔加入100μL稀释好的PE anti-human IgG FC荧光二抗(BioLegend，0.5μL/1×105个细胞配制)，冰上避光孵育30min。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，结果见图5A-5B，可以看出本发明的单抗对HepG2细胞或Hep3B细胞均具有很好的亲和力。
实施例7
流式细胞术检测亲和成熟单抗与肿瘤细胞的结合
应用流式细胞术测定各抗GPC3抗体与HepG2细胞的亲和力。具体操作为：各人源化单抗，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：IgG1、GC90-4mu、GC90LH6、GC90LH7、GC90LH8、GC90LH9、GC90LH10和GC90LH11，工作浓度为：100nM、25nM、6.25nM往下以4为倍数依次稀释共8-9个点。用胰酶消化HepG2细胞，1000rpm离心5min，弃上清，用含FACS Buffer重悬细胞，浓度为2×106个/mL，96孔U底板中每孔加入50μL，再将上述稀释好的抗体每孔加入50μL，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2次。每孔加入100μL稀释好的PE anti-human IgG FC荧光二抗(BioLegend，0.5μL/1×105个细胞配制)，冰上避光孵育30min。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，结果见图5C-5D，可以看出本发明的单抗对HepG2细胞均具有很好的亲和力。
实施例8
流式细胞术检测人源化单抗的内化效率
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：用FACS Buffer将GC90和hYP7HM单抗浓度稀释至1nM实验浓度，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，洗去未结合的抗体后，将细胞转移至37℃分别孵育0、1、2、4h，孵育完成后立即加入100μL预冷FACS buffer终止内化，随后3500rpm离心3min后弃去上清。按照实施例5中方式配置二抗，每孔使用100μL二抗重悬细胞，并置于冰上孵育30min。离心洗涤两次，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，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％，结果见图6，根据图6的结果可以看出，本发明的人源化单抗具有很好的内化效率。
实施例9
抗体-药物偶联物的制备
9.1定点偶联
将抗体重链239位残基突变为半胱氨酸(S239C，EU编号，对应于GC90-4mu重链的第243位)，从而通过该半胱氨酸经由接头与药物进行定点偶联。具体而言，将单抗原液置换为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，获得IgG1-PBD，GC90-4mu-PBD、GC90LH8-PBD、GC90LH9-PBD、GC90LH10-PBD、GC90LH11-PBD以及IgG1-DUBA，GC90-4mu-DUBA、hYP7HM-DUBA定点偶联产物，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。
表7 ADC产物检测分析

9.2随机偶联
将单抗原液置换为20mM NaAc-HAc缓冲液(pH5.5)并调节浓度至5±1mg/mL，按照三(2-羧乙基)膦(TCEP)与抗体的摩尔比20:1，将所需量的TCEP加入反应体系中混匀后于37℃还原反应3小时。还原反应结束后使用10kD超滤离心管离心换液成20mM NaAc/Tris，1mM EDTA，pH7.0缓冲液(3500×g，4次)，调节浓度至5±1mg/mL。向上述反应液中加入适量的DMSO(～10％)，按照LP药物与抗体摩尔比10:1，将所需量的LP溶液(5～10mM，预先溶解在DMSO中)加入反应体系中混匀后于21～25℃偶联反应1小时。然后按照N-乙酰半胱氨酸(NAC)与抗体摩尔比40:1加入NAC溶液混匀后于21～25℃终止反应1小时。终止反应结束后使用10kD超滤离心管换液成20mM His/His-HCl，pH6.0缓冲液(3500×g，4次)，并调节至目标浓度，经0.22μm除菌滤器过滤后，≤-20℃保存。
经偶联反应后，获得IgG1-Dxd，GC90-4mu-Dxd随机偶联产物，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、Huh7肝癌细胞的杀伤作用。具体操作为：HepG2、Hep3B、Huh7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，37℃、5％ CO2培养过夜。再使用10％ FBS+DMEM培养基，将各抗体-药物偶联物稀释至333.5nM，按40μL/孔加入到含160ul/孔的96孔板中5倍稀释，初始浓度为66.7nM，连续倍比稀释8个浓度点，并做重复孔；之后置于37℃、5％CO2恒温培养箱培养。在第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7所示，随着给药浓度升高，本发明的抗体-药物偶联物对各类肝癌细胞显示出良好的细胞杀伤效果。
实施例11
11.1受试物的抗肿瘤试验1
建立人源肝癌Hep3B细胞株皮下异种移植荷瘤裸鼠模型。
收集指数生长期的Hep3B细胞，细胞重悬在1:1的PBS与基质胶中调整细胞密度为5×107，实验小鼠于右前侧背部皮下接种5×106Hep3B细胞(0.1mL/只)，定期观察肿瘤生长情况定期观察肿瘤生长情况；待肿瘤生长至平均体积大于100mm3时根据肿瘤大小随机分组给药，每组6只，分组当天定义为第0天；分组当天分别单次尾静脉注射给予1.5mg/kg的IgG1-PBD、GC90-4mu-PBD。每周进行两次肿瘤体积测量。体积计算公式如下：
肿瘤体积(mm3)＝1/2×(a×b2)(其中a表示长径，b表示短径)
结果：数据表明GC90-4mu-PBD(1.5mg/kg)具有显著的抗肿瘤作用(图8A)。
11.2受试物的抗肿瘤试验2
建立人源肝癌Hep3B细胞株皮下异种移植荷瘤裸鼠模型。
收集指数生长期的Hep3B细胞，细胞重悬在1:1的PBS与基质胶中调整细胞密度为5×107，实验小鼠于右前侧背部皮下接种5×106Hep3B细胞(0.1mL/只)，定期观察肿瘤生长情况定期观察肿瘤生长情况；待肿瘤生长至平均体积大于100mm3时根据肿瘤大小随机分组给药，每组6只，分组当天定义为第0天；分组当天分别单次尾静脉注射给予5或10mg/kg的IgG1-DUBA、hYP7HM-DUBA、GC90-4mu-DUBA。每周进行两次肿瘤体积测量。体积计算公式如下：
肿瘤体积(mm3)＝1/2×(a×b2)(其中a表示长径，b表示短径)
结果：数据表明GC90-4mu-DUBA(5mg/kg)和hYP7HM-DUBA(10mg/kg)均具有显著的抗肿瘤作用(图8B)。
11.3受试物的抗肿瘤试验3
建立人源肝癌Hep3B细胞株皮下异种移植荷瘤裸鼠模型
收集指数生长期的Hep3B细胞，细胞重悬在1:1的PBS与基质胶中调整细胞密度为5×107，实验小鼠于右前侧背部皮下接种5×106Hep3B细胞(0.1mL/只)，定期观察肿瘤生长情况定期观察肿瘤生长情况；待肿瘤生长至平均体积80～150mm3(主实验组)或300～500mm3(卫星组)根据肿瘤大小和小鼠体重随机分组给药，每组6只(卫星组IgG-PBD组1只)，分组第二天定义为第0天(Day 0)：卫星组Day 0单次尾静脉注射给予1.5mg/kg GC90-4mu-PBD、IgG1-PBD，卫星组IgG-PBD组于给药后24h最大量采血收集血清并取瘤，GC90-4mu-PBD组交替采血并取瘤(15min采血清，7h、D1、D2、D3、D5、D7采血清并取瘤)；主实验组Day 0单次尾静脉注射给予IgG1-PBD(0.6mg/kg)、GC90-4mu-PBD(0.1、0.3、0.6mg/kg)、GC90-4mu-Dxd(3、10mg/kg)，每周测量两次小鼠的体重和肿瘤的大小。体积计算公式如下：
肿瘤体积(mm3)＝1/2×(a×b2)(其中a表示长径，b表示短径)
实施例12
GC90抗体的功能表征
12.1种属交叉反应
应用ELISA方法测定GC90对不同种属的GPC3蛋白的结合情况。具体操作为：分别将各GPC3蛋白(6μg/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GC90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)，其工作浓度为：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50μL TMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm测定OD值，结果如表8所示。
表8 GC90与不同种属的GPC3蛋白的结合情况
注：√代表亲和识别，×代表不识别。
12.2识别表位的精细定位
将GPC3-B肽的氨基酸序列分成多个重叠肽，所述重叠肽的氨基酸序列如表9所示，合成重叠肽并使用ELISA测量每种肽与GC90之间的结合特性，以进一步精确鉴定GC90识别的抗原表位。具体操作为：分别将各重叠肽(6μg/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GC90用含1％ BSA的PBST进行系列梯度稀释(阴性对照为1％ BSA)，依次加入96孔板中(100μL/孔)，其工作浓度为：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50μL TMB(SURMOPICS)反应，用1M H2SO4终止反应，酶标仪450nm测定OD值。结果如图9所示，以测量的抗原抗体复合物的OD450值，展示GC90与每种肽之间的结合强度，可见该抗体仅与GPC3-B3或包含GPC3-B3的长片段之间存在强结合活性，可以确定GPC3抗原表位区域位于GPC3-B3，即人GPC3蛋白残基485-496位(DKNLDEEGFESG)。
表9重叠肽的氨基酸序列
进一步地，基于氨基酸序列分析人GPC3蛋白和鼠Gpc3蛋白之间的同源性(图10)，发现在GPC3-B3区域内二者存在2个氨基酸残基的差异(猴GPC3蛋白参考Genbank ID：XP_005594665.1，亦包括GPC3-B3)，即人GPC3蛋白的第487位氨基酸残基(N)和第493位氨基酸残基(F)，结合种属交叉检测结果可知，由于鼠Gpc3蛋白在对应于人GPC3蛋白的第487位天冬酰胺和第493位苯丙氨酸处发生了变异，从而导致GC90无法与鼠Gpc3蛋白结合，表明人GPC3蛋白的第487位和/或第493位是抗原表位区域的核心结合位点之一。
图11汇总了各抗GPC3抗体在GPC3蛋白上的抗原识别区域，可见本发明的抗体所结合的抗原表位远离其他已报道的抗GPC3抗体的抗原结合区域，其识别一种全新的GPC3抗原表位。本发明鉴定了新颖的GPC3抗原表位，与所述表位结合的抗体具备高亲和力和内化效率，采用该GPC3抗原表位制备免疫原，为抗GPC3抗体领域开辟了一个新的研发方向，能够降低抗体制备和筛选的不确定性，以及有利于开发功能改善的新抗体。
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 (25)

  1. 一种抗GPC3抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)；
    所述重链可变区(VH)包含HCDR1、HCDR2和HCDR3区，且所述HCDR1、HCDR2和HCDR3区分别包含与SEQ ID Nos：1-2、6-9任一所示的氨基酸序列的CDR1、CDR2和CDR3区至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；
    或分别包含与SEQ ID Nos：1-2、6-9任一所示的氨基酸序列的CDR1、CDR2和CDR3区相比发生至多3个、2个或1个突变的序列；
    所述轻链可变区(VL)包含LCDR1、LCDR2和LCDR3区，且所述LCDR1、LCDR2和LCDR3区分别包含与氨基酸序列SEQ ID No：3的CDR1、CDR2和CDR3区至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；
    或分别包含与氨基酸序列SEQ ID NO：3的CDR1、CDR2和CDR3区相比发生至多3个、2个或1个突变的序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段具有以下生物学功能：与SEQ ID Nos：37或38所示的抗原特异性结合，并且不结合SEQ ID No：36所示的抗原。
  3. 根据权利要求1所述的抗体或其抗原结合片段，所述突变的位置选自SEQ ID Nos：1-2、6-9任一所示氨基酸序列的第56位(D56)、第100位(Q100)或第102位(S102)中的一个或多个；
    优选地，所述突变选自Q100R、S102R、D56A、D56K+Q100R、D56A+S102R或D56K+S102R。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其特征在于，所述HCDR1、HCDR2和HCDR3区分别具有与SEQ ID Nos：1-2、6-9、23-28任一所示氨基酸序列的CDR1、CDR2和CDR3区相同的序列；以及所述LCDR1、LCDR2和LCDR3区分别包含与氨基酸序列SEQ ID No：3的CDR1、CDR2和CDR3区相同的序列。
  5. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于，所述CDR1、CDR2和CDR3区根据IMGT、Kabat、Chothia、AbM或Contact的方式定义。
  6. 根据权利要求5所述的抗体或其抗原结合片段，其特征在于，所述VH包含与SEQ ID Nos：1-2、6-9、23-28任一所示氨基酸序列至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；和/或
    所述VL包含与SEQ ID No：3至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其特征在于，所述VH包含与氨基酸序列SEQ ID Nos：1-2、6-9、23-28任一相同的序列；所述VL包含与氨基酸序列SEQ ID NO：3相同的序列。
  8. 根据权利要求1-7任一项所述的抗体或其抗原结合片段，其特征在于，其还包含重链恒定区(CH)和轻链恒定区(CL)；优选地，所述CH包含与氨基酸序列SEQ ID Nos：4、19或20相同的序列；所述CL包含与氨基酸序列SEQ ID No：5相同的序列。
  9. 根据权利要求8所述的抗体或其抗原结合片段，其特征在于，所述重链(H)包含与氨基酸序列SEQ ID Nos：11-18、29-34任一至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；和/或所述轻链(L)包含与氨基酸序列SEQ ID No：10至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  10. 根据权利要求9所述的抗体或其抗原结合片段，其特征在于，所述重链(H)包含与氨基酸序列SEQ ID Nos：11-18、29-34任一相同的序列；所述轻链(L)包含与氨基酸序列SEQ ID No：10相同的序列。
  11. 一种抗GPC3抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段结合SEQ ID Nos：37或38所示的抗原，并且不结合SEQ ID No：36所示的抗原；
    或者，所述抗体或其抗原结合片段与参考抗体竞争结合GPC3蛋白的相同表位，所述参考抗体包含SEQ ID NO：11所示的重链以及SEQ ID NO：10所示的轻链；
    优选地，所述抗GPC3抗体或其抗原结合片段能够阻断参考抗体与GPC3蛋白的结合的至少50％、60％、70％、80％、90％、95％或99％。
  12. 一种GPC3抗原表位肽，其特征在于，所述GPC3抗原表位肽由人GPC3蛋白残基485-496内的至少7个连续氨基酸残基组成，所述人GPC3蛋白的氨基酸序列如SEQ ID NO：35所示；并且至少包含第487位的天冬酰胺和第493位的苯丙氨酸中的一个以上，以及所述GPC3抗原表位肽具有如下一种或多种生物学功能：
    (1)与抗GPC3抗体特异性结合；
    (2)在受试者体内诱导针对GPC3的免疫应答(例如体液免疫应答)；
    (3)在受试者体内诱发抗GPC3抗体的产生；
    (4)预防和/或治疗受试者与GPC3相关的疾病。
  13. 根据权利要求12所述的GPC3抗原表位肽，其特征在于，所述GPC3抗原表位肽由SEQ ID NO:38所示的氨基酸序列组成。
  14. 一种重组抗原，其特征在于，所述重组抗原包含权利要求12或13所述的GPC3抗原表位肽和载体蛋白。
  15. 一种嵌合抗原受体，其包含权利要求1-11任一项的抗GPC3抗体或其抗原结合片段。
  16. 一种多核苷酸，其编码权利要求1-11任一项所述抗的GPC3抗体或其抗原结合片段，权利要求12-13任一项所述的GPC3抗原表位肽，或权利要求14所述的重组抗原。
  17. 一种表达载体，其包含权利要求16所述的多核苷酸。
  18. 一种宿主细胞，其包含权利要求16的多核苷酸或权利要求17的表达载体。
  19. 一种抗体-药物偶联物，其特征在于，其包含权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段以及药物或毒素。
  20. 根据权利要求19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。
  21. 一种双特异性或多特异性抗体分子，其特征在于，包含权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段。
  22. 一种药物组合物，其特征在于，其包含权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段，或权利要求12-13任一项所述的GPC3抗原表位肽，或权利要求14所述的重组抗原，或权利要求15所述的嵌合抗原受体，或权利要求16所述的核酸分子，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19-20任一项所述的抗体-药物偶联物，或权利要求21所述的双特异性或多特异性抗体分子；以及一种以上药学上可接受的载体。
  23. 一种试剂盒，其包含权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段，或权利要求12-13任一项所述的GPC3抗原表位肽，或权利要求14所述的重组抗原，或权利要求19-20任一项所述的抗体-药物偶联物，或权利要求21所述的双特异性或多特异性抗体分子。
  24. 权利要求1-11任一项所述的抗GPC3抗体或其抗原结合片段，权利要求12-13任一项所述的GPC3抗原表位肽，权利要求14所述的重组抗原，权利要求15所述的嵌合抗原受体，权利要求16所述的核酸分子，权利要求17所述的载体，权利要求18所述的宿主细胞，权利要求19-20任一项所述的抗体-药物偶联物，权利要求21所述的双特异性或多特异性抗体分子，或权利要求23所述的试剂盒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。
  25. 权利要求12-13任一项所述的GPC3抗原表位肽，或权利要求14所述的重组抗原在如下任一中的应用：
    (1)制备抗GPC3抗体或其抗原结合片段；
    (2)制备用于治疗和/或预防和/或诊断受试者与GPC3相关的疾病的产品；
    (3)制备检测抗GPC3抗体或其抗原结合片段的产品；
    (4)检测抗GPC3抗体或其抗原结合片段；
    (5)筛选抗GPC3抗体或其抗原结合片段。
PCT/CN2024/099094 2023-06-16 2024-06-14 一种抗gpc3抗体或抗原结合片段及其用途 Pending WO2024255815A1 (zh)

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