WO2022268196A1 - 靶向gpc3的抗原结合蛋白 - Google Patents

靶向gpc3的抗原结合蛋白 Download PDF

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WO2022268196A1
WO2022268196A1 PCT/CN2022/101037 CN2022101037W WO2022268196A1 WO 2022268196 A1 WO2022268196 A1 WO 2022268196A1 CN 2022101037 W CN2022101037 W CN 2022101037W WO 2022268196 A1 WO2022268196 A1 WO 2022268196A1
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seq
amino acid
acid sequence
binding protein
sequence shown
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PCT/CN2022/101037
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English (en)
French (fr)
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姜钰超
刘恒
王苗苗
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天辰生物医药(苏州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • A61K39/464495—Prostate specific membrane antigen [PSMA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein and a chimeric antigen receptor capable of specifically binding to GPC3.
  • Glypican3 is a 70kd membrane protein that belongs to the Glypicans family. It is highly and concentratedly expressed during embryonic development and exhibits tissue specificity. After expression, it will be cleaved by furin enzyme to generate the N-terminal 40kd soluble part and the 30kd part anchored by GPI molecules at the C-terminus of the cell membrane. Genomics and functional studies have shown that GPC3 plays an important role in maintaining the activation of the Wnt pathway and the Hedgehogs pathway. For example, GPC3-coupled heparan sulfate molecules can enhance the binding of Wnts to their receptors and play an important role in maintaining the Wnt pathway.
  • GPC3 is expressed in the brain, digestive tract, bladder, gonad and skin and is highly expressed on the surface of hepatocellular carcinoma; the Wnt pathway plays an important role in liver carcinogenesis, for example, 20% of hepatocellular carcinoma ⁇ -Catenin pathway mutation and Frizzled-7 receptor Overexpression, GPC3 may play a role in promoting the occurrence of some hepatocellular carcinomas. Therefore, there is an urgent need to develop therapeutic drugs targeting GPC3.
  • the present application provides an antigen-binding protein targeting GPC3, which can specifically bind GPC3 with higher affinity.
  • the antigen binding protein can bind human GPC3 and murine GPC3.
  • the present application also provides a chimeric antigen receptor (CAR) comprising the antigen-binding protein, and CAR-T cells, and the CAR-T cells have strong killing ability against HepG2 cells.
  • CAR chimeric antigen receptor
  • the application provides an isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO:17.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 11.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12.
  • the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10.
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ ID NO: The amino acid sequence shown in 13.
  • said isolated antigen binding protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises SEQ ID NO: 14 amino acid sequence.
  • said isolated antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises SEQ ID NO: 15 amino acid sequence.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 16.
  • the antigen binding protein of described separation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:13, described H-FR2 comprises the amino acid sequence shown in SEQ ID NO:14, said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:15, and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:16.
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 17.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • said heavy chain constant region is derived from a human IgG constant region.
  • said heavy chain constant region is derived from a human IgG1 constant region.
  • the isolated antigen binding protein comprises at least one CDR in a VL comprising the amino acid sequence set forth in SEQ ID NO:25.
  • the isolated antigen binding protein comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19.
  • the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen-binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 , and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein comprises L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1
  • the L-FR1 comprises SEQ ID NO: The amino acid sequence shown in 21.
  • said isolated antigen binding protein comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises SEQ ID NO:22 amino acid sequence.
  • said isolated antigen binding protein comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises SEQ ID NO:23 amino acid sequence.
  • the isolated antigen binding protein comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: The amino acid sequence shown in 24.
  • the antigen binding protein of described separation comprises L-FR1, L-FR2, L-FR3 and L-FR4, and described L-FR1 comprises the aminoacid sequence shown in SEQ ID NO:21, described L-FR2 comprises the amino acid sequence shown in SEQ ID NO:22, said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:23, and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the isolated antigen binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:25.
  • the isolated antigen binding protein comprises an antibody light chain constant region.
  • said light chain constant region is derived from a human Ig ⁇ constant region.
  • the isolated antigen binding protein comprises VH and VL, the VH comprising the amino acid sequence shown in SEQ ID NO: 17, and the VL comprising the amino acid sequence shown in SEQ ID NO: 25.
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragments include Fab, Fab', Fv fragments, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of specifically binding glypican 3 (GPC3).
  • the GPC3 comprises human GPC3 and/or murine GPC3.
  • the present application also provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, the antigen-binding domain comprising the Isolated antigen binding protein.
  • the antigen binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.
  • the antigen binding domain of the chimeric antigen receptor comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, and the LCDR2 comprises SEQ ID NO The amino acid sequence shown in: 19, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen binding domain of the chimeric antigen receptor comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen binding domain of the chimeric antigen receptor comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:25.
  • the antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:34.
  • said antigen binding domain of said chimeric antigen receptor comprises an antibody or antigen binding fragment thereof.
  • said antigen binding domain of said chimeric antigen receptor comprises a scFv.
  • said transmembrane domain of said chimeric antigen receptor comprises a transmembrane domain derived from a protein selected from the group consisting of alpha, beta or zeta chains of T cell receptors, CD28, CD3e , CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • said transmembrane domain of said chimeric antigen receptor is a CD8 ⁇ transmembrane region.
  • said transmembrane domain of said chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:36.
  • said co-stimulatory domain of said chimeric antigen receptor comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.
  • said co-stimulatory domain of said chimeric antigen receptor is the 4-1BB cytoplasmic region.
  • said co-stimulatory domain of said chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:37.
  • said intracellular signaling domain of said chimeric antigen receptor comprises a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor further comprises a hinge region comprising a CD8 ⁇ hinge region.
  • the hinge region in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:35.
  • the chimeric antigen receptor is also linked to a signal peptide.
  • said signal peptide in said chimeric antigen receptor comprises a CD8 ⁇ signal peptide.
  • the signal peptide in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:33.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:39.
  • the present application also provides an immune effector cell comprising the chimeric antigen receptor.
  • the immune effector cells comprise T cells.
  • the present application also provides a multispecific antigen-binding protein, which comprises the isolated antigen-binding protein.
  • the multispecific antigen binding protein comprises a moiety that targets CD3.
  • the CD3 comprises human CD3.
  • the present application also provides an isolated nucleic acid molecule encoding said isolated antigen binding protein, said chimeric antigen receptor or said multispecific antigen binding protein.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a host cell comprising the nucleic acid molecule or the vector.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein, the chimeric antigen receptor, the immune effector cell or the multispecific antigen binding protein, and optionally pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the isolated antigen-binding protein, the chimeric antigen receptor, the immune effector cell, the multispecific antigen-binding protein or the pharmaceutical composition.
  • the present application also provides the preparation of the isolated antigen binding protein, the chimeric antigen receptor, the immune effector cell, the multispecific antigen binding protein or the pharmaceutical composition
  • the use of said medicine is used for prevention and/or treatment of disease and/or disease.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the present application also provides a kit for detecting the presence and/or content of GPC3 in a sample, the kit comprising the isolated antigen-binding protein, the chimeric antigen receptor, the immune effector cell, the Said multispecific antigen binding protein, said nucleic acid molecule, said carrier, said host cell and/or said pharmaceutical composition.
  • Figure 1 shows the results of reducing SDS-PAGE of the purified antigen-binding protein described in the present application.
  • Figure 2 shows the results of preliminary determination of Jurkat cell phenotype.
  • FIG. 3 shows the detection results of the GPC3 positive rate of target cells HePG2.
  • Figure 4 show the CD69 expression detection of CAR-Jurkat cells obtained by using the antigen binding protein described in this application.
  • Figure 7 shows the detection of CD69 expression in CAR-Jurkat cells obtained using the antigen-binding protein described in this application.
  • Figure 8 shows the detection of the killing effect of the CAR-T described in this application on target cells.
  • GPC3 Glypican 3
  • the GPC3 may include human GPC3, and its sequence information can be found in UniProtKB/Swiss-Prot accession No.P51654.
  • the GPC3 may comprise mouse GPC3.
  • the term also covers homologues, derivatives, variants and functionally active fragments thereof.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • the "isolated antigen binding protein” may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, e.g., to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions, e.g., comprising biocompatible polymers.
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop.
  • antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum.
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • antibody also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind GPC3).
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, GPC3).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) of the H chain (VH).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody.
  • a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment may also include part or all of the heavy chain.
  • the term "F(ab')2" generally refers to antibody fragments produced by pepsin digestion of intact antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen-binding activity and are capable of cross-linking antigen.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions.
  • the heavy and light chain variable regions include, for example, CDRs.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable
  • the regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have the VL and VH variable regions in any order (for example, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include VL-linker-VH or VH-Linker-VL can be included.
  • the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al.
  • monoclonal antibody generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • the modifier "monoclonal” denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies of experimental animals, such as rodents ("parent antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibodies are less likely to elicit an adverse immune response in a human individual than the parent antibody reduced sex.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen.
  • a humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region.
  • a “humanized antibody” retains antigen specificity similar to the original antibody.
  • “Humanized” forms of non-human antibodies can comprise, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.
  • CAR Chimeric Antigen Receptor
  • TAA tumor-associated antigen
  • transmembrane domain (Transmembrane Domain) generally refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.
  • co-stimulatory domain generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens.
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
  • intracellular signaling domain generally refers to the component of CAR located in intracellular signal transduction, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: its It may be selected from the CD3 ⁇ intracellular domain and the 4-1BB intracellular domain.
  • signal peptide generally refers to a short (about 5-30 amino acids in length) peptide chain that directs the transfer of newly synthesized proteins to the secretory pathway.
  • multispecific generally refers to the ability of an antigen binding molecule to specifically bind at least two different antigenic determinants.
  • the term may encompass the concept of bispecificity.
  • bispecific antigen binding proteins comprise two antigen binding sites, each of which is specific for a different antigenic determinant.
  • a bispecific antigen binding protein is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two different cells.
  • nucleic acid molecule generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can transform, transduce or transfect the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but not only.
  • the term "host cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule of the invention or a nucleic acid molecule of the invention. the carrier described.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • Cells may include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells.
  • the term "pharmaceutical composition” generally refers to a composition for preventing/treating a disease or condition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • pharmaceutically acceptable carrier generally includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is effective in the dosage and concentration employed to the cell or mammal to which it is exposed. non-toxic.
  • the term "specifically binds" or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, easier, and/or for a longer duration than it binds other targets .
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • an antigen binding protein is specific for a particular epitope that cross-reacts with multiple antigens, where specific antibodies bind multiple antigens that carry the cross-reactive epitopes.
  • antigen binding protein binding sites and/or antigen binding proteins having specific binding cross-reactive epitopes are also referred to as multispecific or cross-specific binding site antigen binding proteins, respectively.
  • an antigen binding protein may have a multispecific binding site that specifically binds epitopes that cross-react with a plurality of different antigens.
  • protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, substituted, deleted or added one or more A protein or polypeptide of amino acids.
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homologue may be at least about 85% (eg, having at least about 85%) of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to GPC3). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or peptides.
  • the homology generally refers to the similarity, similarity or association between two or more sequences. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST.
  • the term "comprises” generally refers to the meanings of including, encompassing, containing or encompassing. In some cases, it also means “for” and “consisting of”.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In this application, the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the isolated antigen binding protein can be defined by IMGT.
  • the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is identical to the amino acid sequence shown in SEQ ID NO: 17, the HCDR obtained in any form can fall under the protection of the present application. within range.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 13.
  • the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO: 14 amino acid sequence.
  • the isolated antigen-binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO:15 amino acid sequence.
  • the antigen binding protein of described separation can comprise H-FR4, and the N terminal of described H-FR4 is directly or indirectly connected with the C terminal of described HCDR3, and described H-FR4 can comprise SEQ ID NO: The amino acid sequence shown in 16.
  • the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:14, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:15, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:16 amino acid sequence.
  • the isolated antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region can be derived from that of any immunoglobulin, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody heavy chain constant region can be derived from a human IgG heavy chain constant region.
  • the heavy chain constant region of the immunoglobulin may include mutants thereof.
  • the heavy chain constant region of the antibody may be derived from the heavy chain constant region of any one of human IgG1-4.
  • the isolated antigen binding protein may be derived from a human IgGl heavy chain constant region.
  • the isolated antigen-binding protein may comprise at least one CDR in the variable region VL of the antibody light chain, and the VL may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the LCDR of the isolated antigen-binding protein can be divided in any form, as long as the VL is identical to the amino acid sequence shown in SEQ ID NO: 25, the LCDR obtained by dividing in any form can fall into the protection of the present application within range.
  • the isolated antigen-binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen-binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the isolated antigen-binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:19 sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:18.
  • the isolated antigen-binding protein may comprise L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 21.
  • the isolated antigen binding protein may comprise L-FR2, and the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise SEQ ID NO:22 amino acid sequence.
  • the isolated antigen-binding protein may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise SEQ ID NO:23 amino acid sequence.
  • the antigen binding protein of described separation can comprise L-FR4, and the N terminal of described L-FR4 is directly or indirectly connected with the C terminal of described LCDR3, and described L-FR4 can comprise SEQ ID NO: The amino acid sequence shown in 24.
  • the isolated antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:22, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:23, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:24 amino acid sequence.
  • the isolated antigen-binding protein may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the isolated antigen binding protein may comprise an antibody light chain constant region.
  • the light chain constant region can be derived from a human antibody light chain constant region.
  • the light chain constant region may be derived from a human Ig ⁇ constant region.
  • the isolated antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO:17, and the VL may comprise the amino acid sequence shown in SEQ ID NO:25 .
  • the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
  • the isolated antigen-binding protein described in the present application may comprise heavy chain and/or light chain sequences with one or more conservative sequence modifications.
  • conservative sequence modification refers to the amino acid modification that will not significantly affect or change the binding properties of the antibody.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues having similar side chains are known in the art.
  • one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described herein may be replaced with other amino acid residues from the same side chain group.
  • mice can be immunized with GPC3-Fc fusion protein, and mouse splenocytes can be obtained to screen for antigen-binding proteins with GPC3 antigen affinity.
  • Antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.
  • antigen binding proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc. antigen-binding activity.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting e.g., Western blot
  • flow cytometry e.g., FACS
  • immunohistochemistry e.g., immunofluorescence, etc. antigen-binding activity.
  • the isolated antigen binding protein can specifically bind GPC3.
  • the GPC3 may comprise human GPC3.
  • the frozen-thawed supernatant of clones screened for specific binding to the extracellular domain of GPC3 can be analyzed by using an Octet K2 molecular interaction analyzer. The biotinylated GPC3-huIgG1 Fc fusion protein was immobilized on the SA probe, and the frozen-thawed supernatant was used as the analyte for affinity determination.
  • the isolated antigen binding protein can be produced at a rate of less than or equal to about 6 ⁇ 10 ⁇ 3 1/s, less than or equal to about 5.98 ⁇ 10 ⁇ 3 1/s, less than or equal to about 5.95 ⁇ 10 ⁇ 3 1/s, 3 1/s, less than or equal to about 5.94 ⁇ 10 -3 1/s, less than or equal to about 5.92 ⁇ 10 -3 1/s, less than or equal to about 5.90 ⁇ 10 -3 1/s, less than or equal to about 5.8 ⁇ 10 -3 1/s, less than or equal to about 5.72 ⁇ 10 -3 1/s, less than or equal to about 5.71 ⁇ 10 -3 1/s, less than or equal to about 5.70 ⁇ 10 -3 1/s, less than or equal to about 5.50 ⁇ 10 -3 1/s, less than or equal to about 5.40 ⁇ 10 -3 1/s, less than or equal to about 5.34 ⁇ 10 -3 1/s, less than or equal to about 5.30 ⁇ 10 -3 1/s, less than or Equal to
  • the present application also provides a chimeric antigen receptor (CAR), which may comprise an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the antigen-binding domain of the chimeric antigen receptor may comprise the antigen-binding protein described in the present application.
  • the antigen binding domain of the CAR may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11 Amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the antigen binding domain of the CAR may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:19 Amino acid sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen binding domain of the CAR may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 may comprise SEQ ID
  • the amino acid sequence shown in NO:11, the HCDR3 can include the amino acid sequence shown in SEQ ID NO:10, the LCDR1 can include the amino acid sequence shown in SEQ ID NO:20, and the LCDR2 can include the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 19, and the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen-binding domain of the CAR may comprise VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:17.
  • the antigen-binding domain of the CAR may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the antigen binding domain of the CAR may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:17
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:25 .
  • the antigen-binding domain of the CAR may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding domain of the CAR may comprise a single-chain antibody (scFv).
  • the antigen-binding domain of the CAR may target the GPC3 antigen.
  • the transmembrane domain may comprise a transmembrane domain derived from a protein selected from the group consisting of ⁇ , ⁇ or zeta chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain may comprise a CD8 ⁇ transmembrane region. In certain embodiments, the transmembrane domain is the CD8 ⁇ transmembrane region. In certain embodiments, the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the co-stimulatory domain may comprise a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS.
  • the co-stimulatory domain may comprise 4-1BB.
  • the costimulatory domain may be a costimulatory domain of 4-1BB.
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the intracellular signaling domain may comprise a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain is a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:38.
  • the CAR may also include a hinge region.
  • the hinge region may link the antigen binding domain and the transmembrane domain.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:35.
  • the CAR may also include a signal peptide.
  • the signal peptide comprises a CD8 ⁇ signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:33.
  • the CAR may sequentially comprise an antigen-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:34
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the amino acid sequence shown in NO:37, and the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the CAR may sequentially comprise an antigen-binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:34
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:35
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 36, the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 37
  • the intracellular signaling domain may include the amino acid sequence shown in SEQ ID NO: 38.
  • the CAR may sequentially comprise a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:33
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:34
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the costimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:37
  • the intracellular signal transduction A domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the CAR may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the present application also provides immune effector cells, which may comprise the CAR described in the present application.
  • the immune effector cells may comprise T cells.
  • T cells For example, alpha/beta T lymphocytes and gamma/delta lymphocytes.
  • the immune effector cells may include natural killer cells, natural killer T cells, mast cells, and myeloid-derived phagocytes.
  • the present application also provides multispecific antigen-binding proteins.
  • the multispecific antigen-binding protein may comprise a GPC3-targeting portion and the CD3-targeting portion.
  • the GPC3-targeting moiety can comprise any of the isolated antigen binding proteins described herein.
  • the part targeting GPC3 may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.
  • the part targeting GPC3 may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the multispecific antigen binding protein may comprise a bispecific antigen binding protein. In certain embodiments, the multispecific antigen binding protein may comprise a bispecific antibody.
  • the present application also provides an isolated nucleic acid molecule encoding the isolated antigen binding protein or the chimeric antigen receptor described in the present application.
  • the isolated nucleic acid molecule(s) described herein may be nucleotides, deoxyribonucleotides, or ribonucleotides of any length in isolated form, or analogs isolated from their natural environment or artificially synthesized .
  • the present application also provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and Animal viruses, etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • the vector may contain various expression-controlling elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the present application also provides a host cell, which may comprise the nucleic acid molecule or the vector described in the present application.
  • the cells may comprise the immune effector cells described in the present application.
  • the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein, the chimeric antigen receptor, the immune effector cell or the multispecific antigen-binding protein described in the present application, and optionally a pharmaceutically acceptable carrier.
  • the acceptable ingredients of the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, isotonic and absorption delaying agents compatible with the antigen binding protein or the immune effector cells, Usually safe and non-toxic.
  • the present application also provides the preparation of the isolated antigen binding protein, the chimeric antigen receptor, the immune effector cell, the multispecific antigen binding protein, the nucleic acid molecule , the carrier, the host cell, the method of the pharmaceutical composition.
  • the application also provides the isolated antigen binding protein, the chimeric antigen receptor, the immune effector cell, the multispecific antigen binding protein and/or the pharmaceutical composition
  • the purposes in, described medicine is used for preventing and/or treating disease and/or disease.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the immune effector cell, the multispecific antigen binding protein and/or the pharmaceutical composition for use in for the prevention and/or treatment of diseases and/or conditions.
  • the present application also provides a method for preventing and/or treating diseases and/or disorders, the method comprising administering the isolated antigen-binding protein described in the present application, the chimeric antigen to a subject in need receptor, said immune effector cell, said multispecific antigen binding protein and/or said pharmaceutical composition.
  • the diseases and/or disorders include diseases and/or disorders associated with GPC3.
  • the diseases and/or conditions include tumors.
  • the tumor may include solid tumor and/or hematological tumor.
  • the subject may include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the present application also provides a kit for detecting GPC3 in a sample, which may include the isolated antigen-binding protein described in the present application, the chimeric antigen receptor, the immune effector cell, The nucleic acid molecule, the carrier, the cell, the multispecific antigen binding protein and/or the pharmaceutical composition.
  • Example 1 The expression vector construction and eukaryotic expression of the fusion protein (GPC3-huIgG1 Fc) of human phosphatidylinositol proteoglycan 3 (GPC3) and human IgG1 Fc region
  • Chemically synthesized primers containing the gene sequence of the fusion protein signal peptide were used for expression vector construction. Through molecular cloning, the GPC3 gene fragment was spliced with the human IgG1 Fc gene fragment. The spliced product was cloned into pCDNA3.1 (Thermo) using TaKaRa seamless cloning kit.
  • GPC3-huIgG1 Fc fusion protein 2 mg/mL was mixed and emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich) as an antigen, and 10 6-week-old female Balb/c mice were subcutaneously immunized. After the initial immunization, a booster immunization was performed every ten days, and a total of four subcutaneous immunizations were performed. In the fifth immunization, the spleen was directly immunized with MSLN-huIgG1 Fc fusion protein.
  • the GPC3-huIgG1 Fc fusion protein was used for direct intraperitoneal injection for shock immunization, and the mice were sacrificed four days later, and the spleen was removed.
  • Spleen cells were obtained by grinding whole spleens with a cell strainer (BD). After washing twice with PBS, centrifuge at 1000g for 10 minutes to obtain spleen cells. Total RNA was extracted using Trizol RNA extraction kit.
  • the first-strand cDNA was synthesized using the SuperScript TM IV First-Strand Synthesis System Kit.
  • VH-F, VH-R heavy chain variable domain upstream primers and downstream primers
  • VK-F, VK-R light chain variable domain upstream primers and downstream primers
  • the PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, and 72°C final extension for 10 minutes.
  • the amplified VH gene and VK gene were recovered using a DNA gel recovery kit.
  • the equal amount of VH gene and VK gene was mixed as a template, and the scFv gene was amplified by overlapping PCR using the upstream primer scFv-F and the downstream primer scFv-R.
  • the 50uL reaction system add 25uL phusion master mix, 2.5uL (25pmol) of upstream primer, 2.5uL (25pmol) of downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O.
  • the PCR reaction was carried out according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, and 72°C final extension for 10 minutes.
  • the amplified scFv gene fragment was recovered using a DNA gel recovery kit.
  • the scFv gene fragment and the pcomb3XTT vector were digested with SfiI DNA endonuclease, respectively.
  • SfiI DNA endonuclease were digested with SfiI DNA endonuclease, respectively.
  • 50uL reaction system add 2uL of SfiI, 5uL of 10x buffer, 3ug of DNA, and add ddH2O to 50uL. After mixing well, incubate at 50°C for 3 hours.
  • T4 ligase to cyclize the digested scFv gene fragment and digested pcomb3X vector.
  • T4 ligase to cyclize the digested scFv gene fragment and digested pcomb3X vector.
  • T4 ligase 1uL, 10x buffer 5uL, scFv gene 100ng, pComb3X vector 500ng, add ddH2O to 50uL. After mixing well, incubate at 4°C for 16 hours. A small amount of product was taken to verify the ligation efficiency by agarose gel electrophoresis.
  • the GPC3-huIgG1 Fc fusion protein was randomly biotinylated using EZ-Link Sulfo-NHS-LC-Biotin corresponding standard operating procedures.
  • the binding activity of biotinylated GPC3-huIgG1 Fc fusion protein to YP7 chimeric positive control antibody (self-made) was verified by ELISA method.
  • GPC3-huIgG1 Fc fusion protein as the target protein, apply biopanning to the above-mentioned mouse immune antibody library to obtain antibodies that bind to GPC3-huIgG1 Fc fusion protein (especially the extracellular domain of GPC3. Recover the antibody strain library and After growing to the logarithmic phase, use M13KO7 helper phage to rescue the antibody library, resuspend in 2xYT medium containing ampicillin and kanamycin after centrifugation and amplify overnight at 30°C. PEG/NaCl precipitates the phage and dissolves with glycerol/PBST Phage precipitation to obtain immune library phage suspension.
  • Casein-blocked phages were put into casein-blocked biotinylated huIgG1Fc protein (self-made) and casein-blocked Dynabeads M-270 streptavidin co-incubation system, and the supernatant was collected Phage suspension. Further, put the collected phage suspension into the casein-blocked biotinylated GPC3-huIgG1 Fc fusion protein and casein-blocked Dynabeads M-270 streptavidin co-incubation system, wash with PBST Magnetic beads remove phages that cannot bind to the GPC3-huIgG1 Fc fusion protein.
  • the antibody library obtained after the third round of biopanning was diluted and coated on a plate containing ampicillin to obtain a single clone, and the single clone was selected and cultured overnight in a deep well plate. The next day, the deep-well plate was repeatedly frozen and thawed three times in a -20°C refrigerator, and the supernatant was centrifuged for subsequent ELISA reactions. The ELISA reaction was coated with goat anti-human IgG (Fab specific) overnight, and then centrifuged supernatant, biotinylated GPC3-huIgG1 Fc fusion protein and streptavidin-HRP were added in sequence for positive clone screening. This screening step was repeated in two independent experiments to ensure data accuracy.
  • GPC3 ectodomain-specific binding clones were selected by the above two steps.
  • the frozen-thawed supernatants of clones that specifically bind to the extracellular segment of GPC3 screened in the above steps were analyzed using the Octet K2 Molecular Interaction Analyzer.
  • the biotinylated GPC3-huIgG1 Fc fusion protein was immobilized on the SA probe, and the freeze-thawed supernatant was used as the analyte for affinity determination.
  • P3H10 VH amino acid sequence as shown in SEQ ID NO: 1, VL amino acid sequence as shown in SEQ ID NO: 2), P1C7 (VH amino acid sequence as shown in SEQ ID NO: 2) and P1C7 (VH amino acid sequence as shown in SEQ ID NO: 3, the VL amino acid sequence is shown in SEQ ID NO: 4
  • P2G10 the VH amino acid sequence is shown in SEQ ID NO: 5, the VL amino acid sequence is shown in SEQ ID NO: 6
  • P3A8 the VH amino acid sequence is shown in Shown in SEQ ID NO: 7, VL amino acid sequence is shown in SEQ ID NO: 8
  • P3G4 VH amino acid sequence is shown in SEQ ID NO: 9, VL amino acid sequence is shown in SEQ ID NO: 8
  • P3D9 VH amino acid sequence is shown in SEQ ID NO:17, VL amino acid sequence is shown in SEQ ID NO:25
  • P3D9 VH amino acid sequence is shown in SEQ ID NO:17, VL amino
  • scFv antibody A small amount of prokaryotic expression of scFv antibody was carried out by monoclonal bacteria containing phagemids containing the above eight antibody sequences, and the binding ability of scFv antibody to different species of GPC3 was identified by ESLIA. The experiment was repeated twice independently, and ELISA readings were not obtained for the second experiment due to some sample preparation failures.
  • the specific operation steps for the prokaryotic expression of svFv antibody are as follows: Inoculate the monoclonal bacterial liquid into 3mL 2YT/Amp medium according to the inoculation amount of 1 ⁇ , and cultivate overnight at 37°C and 250rpm for activation. The next day, the activated bacterial solution was inoculated into 100mL 2YT/Amp medium according to the inoculum amount of 1%, and cultured at 37°C and 250rmp until the OD600 was 0.5. Add IPTG with a final concentration of 1mM to 10mL 2YT/Amp bacterial liquid, and induce overnight at 30°C and 250rpm.
  • the specific operation steps of scFv antibody binding ELISA reaction with different species of GPC3 are as follows: respectively coat human GPC3-his (self-made) and Mouse GPC3 (self-made) 100ng per well on the ELISA plate, overnight at 4°C; 50ul of each antibody concentrate Mix with 50ul 1X PBST (containing 1% BSA) and add to the ELISA plate as the primary antibody; add the secondary antibody as Goat anti-Mouse IgG F(ab')2Secondary Antibody (1:4000 dilution, the diluent is PBST), 100ul per well; final color reading. It was determined by ELISA that P3A8, P3G4, and P3D9 combined strongly with both human and mouse GPC3. The ELISA results are shown in Table 2.
  • the antibody sequences of P3H10, P1C7, P2G10, P3A8, P3G4, P3D9, P4F12, P1F6 clones and YP7 clones were selected to construct chimeric antigen receptors.
  • YP7 clone Mitsubishi Immunosorbentase
  • the CAR element lentiviral packaging main plasmid containing the following structures from the N-terminus to the C-terminus was constructed by molecular cloning: CD8 ⁇ signal peptide, scFv antibody, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 41BB cytoplasmic region and CD3z cytoplasmic region.
  • Select the clones with correct sequencing inoculate the bacteria solution into 300ml 2YT medium, shake the bacteria overnight and use the plasmid extraction kit (Shanghai Sangong) to complete the extraction of plasmids.
  • the cationic polymer PEI was used to package the lentivirus, and the process was as follows: Dilute the PEI and the third-generation lentivirus packaging plasmid (lentivirus main plasmid, pMD2.G, pMDLg-pRRE, pRSV-Rev) with serum-free DMEM respectively; Add PEI/DMEM to the plasmid/DMEM mixture, vortex to mix, and let stand at room temperature for 15 minutes; add the plasmid-PEI complex to the pre-plated 293T cells.
  • the third-generation lentivirus packaging plasmid lentivirus main plasmid, pMD2.G, pMDLg-pRRE, pRSV-Rev
  • the biological titer of the virus fluid refers to the number of infectious virus particles per milliliter. Accurate determination of the viral infection titer is helpful for the preparation of chimeric antigen receptor-Jurkat cells with the expected positive rate.
  • Take a 24-well culture plate add a gradient dilution of the virus stock solution to each well, the initial infection volume is 1ml, three-fold ratio, five gradient dilutions, the volume is less than 1ml, add 100 ⁇ l containing 1 to each well after supplementing 1ml with the medium ⁇ 10 5 cell suspensions of 293T cells (containing polybrene, the final concentration is 8 ⁇ g/ml), and the infection efficiency of the cells was detected by flow cytometry after 3 days, and the fluorescent dye-labeled protein FITC-Labeled Human Glypican 3 (ACRO biosystem) to stain the cells, and uninfected synchronously cultured 293T cells were used as a negative control group (293T).
  • Plasmid ID package volume Lentivirus titer Total lentivirus P1F6 50ml 2.02E8 TU/ml 4.00E7 TU (0.2ml) P2G10 50ml 3.60E8 TU/ml 7.21E7 TU (0.2ml) P1C7 50ml 1.90E8 TU/ml 3.81E7 TU (0.2ml) P3H10 50ml 6.17E7 TU/ml 1.234E6 TU(0.2ml) P3D9 50ml 2.99E8 TU/ml 5.97E7 TU (0.2ml) P3A8 50ml 3.78E8 TU/ml 7.56E7 TU (0.2ml) P3G4 50ml 3.59E8 TU/ml 7.18E7 TU (0.2ml) P4F12 50ml 1.38E8 TU/ml 2.76E7 TU (0.2ml) YP7 50ml 8.30E8 TU/ml 1.66
  • the specific process is as follows: collect 400ul cells into 1.5ml centrifuge tubes, and add 1000ul PBS to each tube. Centrifuge at 1500rpm for 5min, discard the supernatant, leave about 50ul of liquid in the tube, flick to resuspend the cells. Add 1ml PBS to each tube to resuspend the cells, centrifuge at 1500rpm for 5min, discard the supernatant, and flick to resuspend the cells.
  • the positive rate of YP7-CAR Jurkat cells was 86.34%, the positive rate of P2G10-CAR Jurkat cells was 13.88%, the positive rate of P3G4-CAR Jurkat cells was 43.70%, the positive rate of P1F6-CAR Jurkat cells was 37.95%, and the positive rate of P1C7-CAR Jurkat cells
  • the positive rate of P3H10-CAR Jurkat cells was 88.99%
  • the positive rate of P3D9-CAR Jurkat cells was 82.99%
  • the positive rate of P3A8-CAR Jurkat cells was 86.32%
  • the positive rate of P4F12-CAR Jurkat cells was 30.11%.
  • the GPC3-positive HePG2 cell line was selected as the target cell.
  • detect the phenotype of HePG2 cells (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) by flow cytometry, recover HePG2 cells from the liquid nitrogen tank and culture HePG2 cells, collect HePG2 cells into a 1.5ml centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, and About 50ul of liquid remained in the medium, flick to resuspend the cells.
  • Add 1ml PBS to each tube to resuspend the cells, centrifuge at 1500rpm for 5min, discard the supernatant, and flick to resuspend the cells.
  • the detailed operation steps for the detection of CD69 expression on the surface of Jurkat cells are as follows: collect the cells into 1.5ml centrifuge tubes, and add 1000ul PBS to each tube. Centrifuge at 1500rpm for 5min, discard the supernatant, flick to resuspend the cells. Add 1ml PBS to each tube to resuspend the cells, centrifuge at 1500rpm for 5min, discard the supernatant, flick to resuspend the cells. After adding 200ul PBS to each tube to resuspend the cells, add 1ul APC anti-human CD69 antibody, and stain at room temperature for 10min in the dark.
  • a batch of YP7 CAR-jurkat cells (positive rate 70.48%) and P3D9 CAR-jurkat cells (positive rate 52.72%) were prepared again using the same method as above.
  • the positive rate of the control YP7 CAR-jurkat cells was adjusted to 52.72% with wild-type jurkat cells, and the co-incubation of the effector-target cells was completed under the condition of an effector-target ratio of 10:1.
  • the expression of CD69 on the surface of Jurkat cells in the two groups is shown in Figure 7.
  • the lentivirus required for target cell construction by chemical synthesis, synthesize the luciferase and green fluorescent protein genes connected in series with an internal ribosome entry site (IRES).
  • the lentiviral packaging master plasmid (PCCL-LUC-GFP) was constructed by molecular cloning and the lentivirus was packaged.
  • the HepG2-luc-GFP single cell clone with high expression of green fluorescent protein was screened out with green fluorescent protein as a marker, and expanded and cultured as target cells for CAR-T killing experiments .
  • Anti-human GPC3 chimeric antigen receptor-T cells kill target cells
  • the CAR-T killing assay evaluates the in vitro function of CAR-T cells by detecting the killing effect of CAR-T cells on target cells in vitro.
  • T cells were co-cultured with HepG2-LUC-GFP target cells with different effector-target ratios (based on 3E4 target cells, effector cells were twice, five times and ten times the number of target cells), and target cells and target cells were set at the same time.
  • Negative control group (NT) mixed with T cells not transfected with CAR elements. The maximum release volume hole was set in the experiment. At the end of the experiment, the cells were lysed by repeated freezing and thawing, and the fluorescence value of the cell supernatant was taken to detect the maximum release volume fluorescence value.
  • the experimental groups and results are shown in Figure 8.
  • the experimental data showed that at each effect-to-target ratio, the killing rate of P3D9-CAR-T cells on target cells was higher than that of the positive control YP7-CAR-T cells, while the effect-to-target ratio of P3H10-CAR-T cells was 5:1 The killing rate was lower than the positive control YP7-CAR-T cells at 10:1.
  • the antibody sequences described in the table below were constructed in the pTT5 eukaryotic expression vector, transfected into HEK293 cells, and the supernatant was collected after 6 days of expression and purified by protein A affinity chromatography. SEC-HPLC detection shows that the monomer content of the two antibodies is about 90%, which can be used to detect the activation ability of T cell signal.
  • PSMA prostate-specific membrane antigen
  • the pGL4.3[luc2P/NFAT-RE] (Promega) plasmid was transfected into Jurkat cells and screened with 200ug/ml hygromycin under pressure. After long-term culture, the monoclonal cell line was selected by a flow sorter (SONY SH800) as Jurkat-NFAT luc reporter cells.
  • the qualitative analysis of bispecific antibody activity is planned to use three concentration gradients with final concentrations of 1E4pM, 1E3pM, and 0pM for the test, and the 1640 complete medium is pre-configured to two concentrations of 5E4pM and 5E3pM.
  • each gradient antibody-containing medium was added to the 96-well plate in a volume of 20ul per well (the medium was added to the 0pM group), mixed and incubated for five hours.
  • the fluorescence signal increased with the increase of the antibody concentration It was proved that the double antibody can effectively stimulate Jurkat T cells, and no increase of fluorescence signal was observed in each concentration gradient in the YP7 double antibody group, indicating that the antibody structure could not effectively stimulate Jurkat T cells at the stated concentration.

Abstract

涉及一种分离的抗原结合蛋白，其包含分离的抗原结合蛋白，所述分离的抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:17所示的氨基酸序列。所述的分离的抗原结合蛋白能够特异性结合GPC3。还提供了所述抗原结合蛋白的制备方法及用途。

Description

靶向GPC3的抗原结合蛋白 技术领域
本申请涉及生物医药领域，具体的涉及一种能够特异性结合GPC3的抗原结合蛋白及嵌合抗原受体。
背景技术
Glypican3(GPC3)是一种70kd膜蛋白，属于Glypicans家族，高度且集中表达于胚胎发育期并且呈现组织特异性，表达之后会被furin酶切割产生N端40kd可溶性部分以及30kd部分通过GPI分子锚定于细胞膜的C端。基因组学和功能研究表明，GPC3对于维持Wnt通路、Hedgehogs通路的激活中有重要作用，例如GPC3偶联的硫酸乙酰肝素分子可以增强Wnts与其受体的结合从而对于维持Wnt通路有重要作用。GPC3表达于脑、消化道、膀胱，性腺和皮肤并高度表达于肝细胞癌表面；肝癌发生中Wnt通路起到了重要的作用，例如20％肝细胞癌β-Catenin通路突变以及Frizzled-7受体过度表达，GPC3在部分肝细胞癌发生过程中可能起到了促进作用。因此，亟需开发针对GPC3的治疗药物。
发明内容
本申请提供了一种靶向GPC3的抗原结合蛋白，其能够以较高亲和力特异性结合GPC3。所述抗原结合蛋白能结合人GPC3和鼠GPC3。另一方面，本申请还提供了包含所述抗原结合蛋白的嵌合抗原受体(CAR)，及CAR-T细胞，所述CAR-T细胞对HepG2细胞具有较强杀伤。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨 基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:13所示的氨基酸序列，所述H-FR2包含SEQ ID NO:14所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区。
在某些实施方式中，在所述分离的抗原结合蛋白中，所述重链恒定区源自人IgG恒定区。
在某些实施方式中，在所述分离的抗原结合蛋白中，所述重链恒定区源自人IgG1恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包含VL中的至少一个CDR，所述VL包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2包含SEQ ID NO:22所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3包含SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR4，所述L-FR4的N端与所述LCDR3的C端直接或间接相连，且所述L-FR4包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:21所示的氨基酸序列，所述L-FR2包含SEQ ID NO:22所示的氨基酸序列，所述L-FR3包含SEQ ID NO:23所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VL，所述VL包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链恒定区。
在某些实施方式中，在所述分离的抗原结合蛋白中，所述轻链恒定区源自人Igκ恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包含VH和VL，所述VH包含SEQ ID NO:17所示的氨基酸序列，且所述VL包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合磷脂酰肌醇蛋白多糖3(GPC3)。
在某些实施方式中，所述GPC3包含人GPC3和/或鼠GPC3。
另一方面，本申请还提供了一种嵌合抗原受体，其包含抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述抗原结合结构域包含所述分离的抗原结合蛋白。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含HCDR1，HCDR2和 HCDR3，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，所述嵌合抗原受体的所述抗原结合结构域包含scFv。
在某些实施方式中，所述嵌合抗原受体的所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在某些实施方式中，所述嵌合抗原受体的所述跨膜结构域为CD8α跨膜区。
在某些实施方式中，所述嵌合抗原受体的所述跨膜结构域包含SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述共刺激域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
在某些实施方式中，所述嵌合抗原受体的所述共刺激域为4-1BB胞质区。
在某些实施方式中，所述嵌合抗原受体的所述共刺激域包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导结构域包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含铰链区，所述铰链区包含CD8α铰链区。
在某些实施方式中，所述嵌合抗原受体中所述铰链区包含SEQ ID NO:35所示的氨基酸 序列。
在某些实施方式中，所述嵌合抗原受体还连接信号肽。
在某些实施方式中，所述嵌合抗原受体中所述信号肽包含CD8α信号肽。
在某些实施方式中，所述嵌合抗原受体中所述信号肽包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:39所示的氨基酸序列。
另一方面，本申请还提供了一种免疫效应细胞，其包含所述嵌合抗原受体。
在某些实施方式中，所述免疫效应细胞包含T细胞。
另一方面，本申请还提供了一种多特异性抗原结合蛋白，其包含所述分离的抗原结合蛋白。
在某些实施方式中，所述多特异性抗原结合蛋白包含靶向CD3的部分。
在某些实施方式中，所述CD3包含人CD3。
另一方面，本申请还提供了分离的核酸分子，其编码所述分离的抗原结合蛋白、所述嵌合抗原受体或所述多特异性抗原结合蛋白。
另一方面，本申请还提供了载体，其包含所述核酸分子。
另一方面，本申请还提供了宿主细胞，其包含所述核酸分子或所述载体。
另一方面，本申请还提供了药物组合物，其包含所述分离的抗原结合蛋白，所述嵌合抗原受体，所述免疫效应细胞或所述多特异性抗原结合蛋白，以及任选地药学上可接受的载剂。
另一方面，本申请还提供了制备所述分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞、所述多特异性抗原结合蛋白或所述药物组合物的方法。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞、所述多特异性性抗原结合蛋白或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包含肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
另一方面，本申请还提供了检测样品中GPC3存在和/或含量的试剂盒，所述试剂盒包括所述分离的抗原结合蛋白，所述嵌合抗原受体，所述免疫效应细胞，所述多特异性抗原结合蛋白，所述核酸分子，所述载体，所述宿主细胞和/或所述药物组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本 申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是纯化后的本申请所述抗原结合蛋白的还原SDS-PAGE结果。
图2显示的是Jurkat细胞表型初步确定结果。
图3显示的是靶细胞HePG2的GPC3阳性率检测结果。
图4，图5和图6显示的是使用本申请所述的抗原结合蛋白得到的CAR-Jurkat细胞的CD69表达检测。
图7显示的是使用本申请所述的抗原结合蛋白得到的CAR-Jurkat细胞的CD69表达检测。
图8显示的是本申请所述CAR-T对靶细胞的杀伤作用检测。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“GPC3(Glypican3)”通常指磷脂酰肌醇蛋白多糖3。在本申请中，所述GPC3可以包含人GPC3，其序列信息可以见UniProtKB/Swiss-Prot accession No.P51654。在本申请中，所述GPC3可以包含鼠GPC3。在本申请中，该术语还涵盖其同源物、衍生物、变体及功能活性片段。
在本申请中，术语“分离的抗原结合蛋白”通常指脱离了其天然存在状态的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工框架区。此类框架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突 变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、AbM、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合GPC3)的其它抗体片段。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，GPC3)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(VH)的第一恒定区(CH1)组成。在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有。在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够 交联抗原。在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。
在本申请中，术语“单克隆抗体”通常指单分子组成的抗体分子制备物。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体的CDR区以外的部分或全部氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修 饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是抗原结合蛋白的可变区和T细胞信号分子的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其包括肿瘤相关抗原(tumor-associated antigen，TAA)结合区、跨膜结构域、共刺激结构域和胞内信号结构域。
在本申请中，术语“跨膜结构域”(Transmembrane Domain)通常是指CAR中穿过细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，术语“胞内信号传导结构域”通常是指CAR位于细胞内信号传导的组分，其包含信号传导结构域和特异性结合所述受体组分的结构域，例如：其可选自CD3ζ胞内域和4-1BB胞内域。
在本申请中，术语“信号肽”(Signal peptide)通常是指引导新合成的蛋白质向分泌通路转移的短(长度约5-30个氨基酸)肽链。
术语“多特异性”通常指抗原结合分子能够特异性结合至少两种不同的抗原性决定簇。在本申请中，该术语可以涵盖双特异性的概念。通常，双特异性抗原结合蛋白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，双特异性抗原结合蛋白能够同时结合两种抗原性决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
在本申请中，术语“核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“宿主细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可 以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞。
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载剂、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更长的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。该术语也适用于，如抗原结合蛋白对与多个抗原交叉反应的特定表位是有特异性的，其中，特异性的抗体能结合携带交叉反应表位的多种抗原。这种抗原结合蛋白的结合位点和/或具有特异性结合交叉反应表位的抗原结合蛋白，分别也被称为多特异性或交叉特异性结合位点的抗原结合蛋白。例如，抗原结合蛋白可具有与多个不同抗原交叉反应的表位特异性结合的多特异性结合位点。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合GPC3的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述 多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合GPC3的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。
在本申请中，术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
分离的抗原结合蛋白
抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在本申请中，所述分离的抗原结合蛋白可以通过IMGT定义。
一方面，本申请提供一种分离的抗原结合蛋白，其可以包含抗体重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:17所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的HCDR可以通过任何形式划分，只要VH与SEQ ID NO:17所示的氨基酸序列相同，以任何形式划分得到的HCDR都可落入本申请的保护范围内。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，所述HCDR1可以包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:15所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N端与所述HCDR3的C末端直接或间接相连，且所述H-FR4可以包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可以包含SEQ ID NO:13所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:14所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VH，所述VH可以包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体重链恒定区。例如，所述抗体重链恒定区可以源自任一种免疫球蛋白的重链恒定区，包括IgM、IgD、IgG、IgA和IgE。例如， 所述抗体重链恒定区可以源自人IgG重链恒定区。在本申请中，所述免疫球蛋白的重链恒定区可以包含其突变体。在本申请中，所述抗体重链恒定区可以源自人IgG1-4中任一种的重链恒定区。例如，所述分离的抗原结合蛋白可以源自人IgG1重链恒定区。
在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:25所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:25所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，所述LCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR2，所述LCDR2可以包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，所述LCDR1可以包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1可以包含SEQ ID NO:21所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2可以包含SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3可以包含SEQ ID NO:23所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4可以包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1可以包含SEQ ID NO:21所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:22所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:23所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VL，所述VL可以包含SEQ ID NO:25 所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链恒定区。例如，所述轻链恒定区可以源自人抗体轻链恒定区。例如，所述轻链恒定区可以源自人Igκ恒定区。
在本申请中，所述分离的抗原结合蛋白可以包含VH和VL，所述VH可以包含SEQ ID NO:17所示的氨基酸序列，且所述VL可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体或其抗原结合片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。在本申请中，所述抗体可以包括单克隆抗体、嵌合抗体、人源化抗体和/或全人源抗体。
此外，需要说明的是，本申请所述分离的抗原结合蛋白可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述分离的抗原结合蛋白中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。在某些实施方式中，本申请所述分离的抗原结合蛋白的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。
在本申请中，可以通过使用GPC3-Fc融合蛋白对小鼠进行免疫，获取小鼠脾细胞筛选具有GPC3抗原亲和力的抗原结合蛋白。
可以通过本领域已知的各种测定鉴别、筛选或表征本申请所述的抗原结合蛋白。
例如，可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白的抗原结合活性。
在本申请中，所述分离的抗原结合蛋白能够特异性结合GPC3。在本申请中，所述GPC3可以包含人GPC3。在某些实施方式中，可以利用Octet K2分子互作分析仪对筛选到GPC3胞外段特异结合克隆的冻融上清进行分析。将生物素化的GPC3-huIgG1 Fc融合蛋白固化在SA探针上，以冻融上清作为分析物，进行亲和力测定。在某些实施方式中，所述分离的抗原结合蛋白可以以小于或等于约6×10 -31/s、小于或等于约5.98×10 -31/s、小于或等于约5.95×10 - 31/s、小于或等于约5.94×10 -31/s、小于或等于约5.92×10 -31/s、小于或等于约5.90×10 -31/s、小于或等于约5.8×10 -31/s、小于或等于约5.72×10 -31/s、小于或等于约5.71×10 -31/s、小于或等于约5.70×10 -31/s、小于或等于约5.50×10 -31/s、小于或等于约5.40×10 -31/s、小于或等于 约5.34×10 -31/s、小于或等于约5.30×10 -31/s、小于或等于约5.28×10 -31/s、小于或等于约5.20×10 -31/s、小于或等于约5.13×10 -31/s、小于或等于约5.03×10 -31/s、小于或等于约5.00×10 - 31/s、小于或等于约4.94×10 -31/s、小于或等于约4.90×10 -31/s、小于或等于约4.80×10 -31/s、小于或等于约4.70×10 -31/s、小于或等于约4.67×10 -31/s、小于或等于约4.60×10 -31/s、小于或等于约4.59×10 -31/s、小于或等于约4.50×10 -31/s、小于或等于约4.40×10 -31/s、小于或等于约4.30×10 -31/s、小于或等于约4.29×10 -31/s、小于或等于约4.10×10 -31/s、小于或等于约4.09×10 -31/s、小于或等于约4.00×10 -31/s、小于或等于约3.90×10 -31/s、小于或等于约3.50×10 -31/s、小于或等于约3.00×10 -31/s、小于或等于约2.50×10 -31/s、小于或等于约2.00×10 - 31/s、小于或等于约1.98×10 -31/s、小于或等于约1.90×10 -31/s、小于或等于约1.85×10 -31/s、小于或等于约1.5×10 -31/s、小于或等于约1.00×10 -31/s的解离常数与所述GPC3结合。
嵌合抗原受体和免疫效应细胞
另一方面，本申请还提供了一种嵌合抗原受体(CAR)，所述CAR可以包含抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导域。
在本申请中，所述嵌合抗原受体的抗原结合结构域可以包含本申请所述的抗原结合蛋白。
在本申请中，所述CAR的抗原结合结构域可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含LCDR1，LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述HCDR1可以包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含VH，所述VH包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含VL，所述VL可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含VH和VL，所述VH可以包含SEQ ID NO:17所示的氨基酸序列且所述VL可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包含抗体或其抗原结合片段。
在本申请中，所述CAR的抗原结合结构域可以包含单链抗体(scFv)。
在本申请中，所述CAR的抗原结合结构域可以靶向GPC3抗原。
在本申请中，所述跨膜结构域可以包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在本申请中，所述跨膜结构域可以包含CD8α跨膜区。在某些实施方式中，所述跨膜结构域为CD8α跨膜区。在某些实施方式中，所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述共刺激结构域可以包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
在本申请中，所述共刺激结构域可以包含4-1BB。在某些实施方式中，所述共刺激结构域可以为4-1BB的共刺激结构域。在某些实施方式中，所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述胞内信号传导域可以包含来自CD3ζ的信号传导结构域。在某些实施方式中，所述胞内信号传导域为来自CD3ζ的信号传导结构域。在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述CAR还可以包含铰链区。在某些实施方式中，所述铰链区可以连接所述抗原结合结构域和所述跨膜结构域。在某些实施方式中，所述铰链区包含SEQ ID NO:35所示的氨基酸序列。
在本申请中，所述CAR还可以包含信号肽。在某些实施方式中，所述信号肽包含CD8α信号肽。在某些实施方式中，所述信号肽包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述CAR可以从N端到C端依次包含抗原结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导域。例如，所述抗原结合结构域可以包含SEQ ID NO:34所示的氨基酸序列，所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列，所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列，且所述胞内信号传导域可以包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述CAR可以从N端到C端依次包含抗原结合结构域、铰链区、跨膜结 构域、共刺激结构域、以及胞内信号传导域。例如，所述抗原结合结构域可以包含SEQ ID NO:34所示的氨基酸序列，所述铰链区可以包含SEQ ID NO:35所示的氨基酸序列，所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列，所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列，且所述胞内信号传导域可以包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述CAR可以从N端到C端依次包含信号肽、抗原结合结构域、铰链区、跨膜结构域、共刺激结构域、以及胞内信号传导域。例如，所述信号肽可以包含SEQ ID NO:33所示的氨基酸序列，所述抗原结合结构域可以包含SEQ ID NO:34所示的氨基酸序列，所述铰链区可以包含SEQ ID NO:35所示的氨基酸序列，所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列，所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列，且所述胞内信号传导域可以包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述CAR可以包含SEQ ID NO:39所示的氨基酸序列。
另一方面，本申请还提供了免疫效应细胞，其可以包含本申请所述的CAR。在本申请中，所述免疫效应细胞可以包含T细胞。例如，α/β的T淋巴细胞和γ/δ淋巴细胞。在某些实施方式中，所述免疫效应细胞可以包括自然杀伤细胞、自然杀伤T细胞、肥大细胞和骨髓源性吞噬细胞。
多特异性抗原结合蛋白
另一方面，本申请还提供了多特异性抗原结合蛋白。在本申请中，所述多特异性抗原结合蛋白可以包含靶向GPC3的部分和所述靶向CD3的部分。例如，所述靶向GPC3的部分可以包含本申请所述的任一分离的抗原结合蛋白。例如，所述靶向GPC3的部分可以包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。例如，所述靶向GPC3的部分可以包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。在某些实施方式中，所述多特异性抗原结合蛋白可以包含双特异性抗原结合蛋白。在某些实施方式中，所述多特异性抗原结合蛋白可以包含双特异性抗体。
核酸分子、载体、细胞、药物组合物、制备方法
另一方面，本申请还提供了分离的核酸分子，其编码本申请所述的分离的抗原结合蛋白或所述嵌合抗原受体。本申请所述的分离的一种或多种核酸分子可以为任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请还提供了宿主细胞，其可以包含本申请所述的核酸分子或所述载体。在本申请中，所述细胞可以包含本申请所述的免疫效应细胞。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞或所述多特异性抗原结合蛋白，以及任选地药学上可接受的载剂。在某些实施方式中，所述药物组合物的可接受成分在所用剂量和浓度下对接收者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。在某些实施方式中，所述药学上可接受的载剂可以包括与所述抗原结合蛋白或所述免疫效应细胞相容的任何和所有的溶剂、分散介质、等渗剂和吸收延迟剂，通常安全、无毒。
另一方面，本申请还提供了制备本申请所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述的免疫效应细胞、所述多特异性抗原结合蛋白、所述核酸分子、所述载体、所述宿主细胞、所述药物组合物的方法。
用途和应用
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞、所述多特异性抗原结合蛋白和/或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞、所述多特异性抗原结合蛋白和/或所述药物组合物，其用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白、所述嵌合抗原受体、所述免疫效应细胞、 所述多特异性抗原结合蛋白和/或所述药物组合物。
在本申请中，所述疾病和/或病症包括与GPC3相关的疾病和/或病症。
在本申请中，所述疾病和/或病症包括肿瘤。
在本申请中，所述肿瘤可以包括实体瘤和/或血液瘤。
在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
另一方面，本申请还提供了检测样品中GPC3的试剂盒，所述试剂盒可以包括本申请所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述的免疫效应细胞、所述的核酸分子、所述的载体、所述的细胞、所述多特异性抗原结合蛋白和/或所述的药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、嵌合抗原受体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 人磷脂酰肌醇蛋白多糖3(GPC3)与人IgG1 Fc区的融合蛋白(GPC3-huIgG1 Fc)的表达载体构建与真核表达
1.合成GPC3 25位至599位氨基酸区间的基因序列及GPC3-huIgG1 Fc融合蛋白的表达载体构建
通过化学合成的方式合成人磷脂酰肌醇蛋白多糖3(GPC3)(UniProtKB/Swiss-Prot accession No.P51654)的第25位谷氨酰胺至第559位组氨酸区间的基因序列，该基因序列编码SEQ ID NO:30所示的氨基酸序列。通过化学合成的方式合成人IgG1重链恒定区(UniProtKB/Swiss-Prot accession No.P01857.1)的第99位谷氨酸至第330位赖氨酸氨酸区间的基因序列，该基因序列编码SEQ ID NO:31所示的氨基酸序列。化学合成含融合蛋白信号肽(SEQ ID NO:32所示的氨基酸序列)基因序列的引物用于表达载体构建。通过分子克隆，将GPC3基因片段与人IgG1 Fc基因片段进行拼接。拼接产物用TaKaRa无缝克隆试剂盒克隆到pCDNA3.1(Thermo)中。
2.重组GPC3-huIgG1 Fc融合蛋白的表达与纯化
以此表达载体转染293T细胞(ATCC)5天后，收集培养上清，用AKTA avant(GE)纯化重组GPC3-huIgG1 Fc融合蛋白。由于糖基化修饰等原因，重组GPC3-huIgG1 Fc融合蛋白经还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约100-120k道尔顿左右，结果如图1所示。
实施例2 抗人GPC3鼠源抗体的制备
1.免疫动物
将2mg/mL的GPC3-huIgG1 Fc融合蛋白作为抗原与等体积的完全弗氏佐剂(Sigma-Aldrich)混合乳化，取10只6周大雌性Balb/c小鼠进行皮下免疫。在初次免疫后，每十天时间进行一次加强免疫，共执行四次皮下免疫，第五次免疫时直接用MSLN-huIgG1 Fc融合蛋白进行脾脏冲击免疫。
2.血清效价检测
每次加强免疫前尾静脉取血50uL，离心去除细胞，保留血清。ELISA微孔板中加入重组GPC3 50ng/孔，4℃包被过夜。PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。加入梯度稀释的小鼠血清，37℃结合1小时。PBST清洗三遍，加入HRP-山羊抗小鼠IgG37℃结合1小时。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值。
3.构建免疫文库
3.1小鼠脾细胞总cDNA获取
利用GPC3-huIgG1 Fc融合蛋白直接进行腹腔注射方式进行冲击免疫，四天后处死小鼠，取脾脏。用细胞筛网(BD)研磨整个脾脏，获取脾脏细胞。用PBS冲洗两遍后，1000g离心10分钟，获取脾脏细胞。使用Trizol RNA提取试剂盒抽提总RNA。
以所述RNA为模板，使用SuperScript TM IV First-Strand Synthesis System试剂盒合成第一链cDNA。
3.2抗体基因扩增与轻重链拼接
以所述cDNA为模板，使用重链可变域上游引物和下游引物(VH-F、VH-R)PCR扩增重链可变域基因，使用轻链可变域上游引物和下游引物引物(VK-F、VK-R)PCR扩增卡帕链可变域基因。在50uL反应体系中，分别加入25uL phusion master mix，上游引物2.5uL(25pmol)，下游引物2.5uL(25pmol)，1.5uL DMSO，0.5uL cDNA和18uL ddH2O。按以下程序进行PCR反应：98℃预变性1分钟后进入温度循环，98℃变性30秒，58℃退火30秒，72℃延伸1分钟，循环30次，72℃最终延伸10分钟。
使用DNA胶回收试剂盒回收扩增得到的VH基因和VK基因。将等量的VH基因和VK基因混合后为模板，利用上游引物scFv-F和下游引物scFv-R通过重叠PCR扩增scFv基因。在50uL反应体系中，分别加入25uL phusion master mix，上游引物2.5uL(25pmol)，下游引物2.5uL(25pmol)，1.5uL DMSO，0.5uL cDNA和18uL ddH2O。按以下程序进行PCR反应：98℃预 变性1分钟后进入温度循环，98℃变性30秒，58℃退火30秒，72℃延伸1分钟，循环30次，72℃最终延伸10分钟。
使用DNA胶回收试剂盒回收扩增得到的scFv基因片段。
3.3构建免疫文库
分别使用SfiI DNA内切酶消化scFv基因片段和pcomb3XTT载体(美国Scripps研究所)。在50uL反应体系中，分别加入SfiI 2uL，10x缓冲液5uL，DNA 3ug，加ddH2O至50uL。充分混匀后，50℃孵育3小时。
使用DNA胶回收试剂盒回收酶切后的scFv基因片段和pcomb3X载体。使用T4连接酶环化酶切后的scFv基因片段和酶切后的pcomb3X载体。在50uL反应体系中，分别加入T4连接酶1uL，10x缓冲液5uL，scFv基因100ng，pComb3X载体500ng，加ddH2O至50uL。充分混匀后，4℃孵育16小时。取少量产物通过琼脂糖凝胶电泳验证连接效率。
将10uL上述连接环化产物加入自制的TG1电转化感受态中，然后使用电转仪进行电击转化。取出10ul电转化后的细菌通过合理的稀释并在含有氨苄青霉素的平板上划线，以此计数并统计噬菌体抗体文库的大小。剩余的电转化后的细菌加入含100ug/mL氨苄青霉素和2％葡萄糖的2xYT培养基，置于加热培养箱培养。培养结束后在4℃以4000G离心10分钟，在沉淀菌中补充适量甘油储存于-80℃作为抗体菌种库。通过多次电转化积累获得scFv免疫文库。
4.鼠源免疫抗体噬菌体文库的筛选与鉴定
4.1 GPC3-huIgG1 Fc融合蛋白的生物素化
应用EZ-Link Sulfo-NHS-LC-Biotin对应标准操作程序对GPC3-huIgG1 Fc融合蛋白进行随机生物素化。用ELISA方法验证生物素化的GPC3-huIgG1 Fc融合蛋白与YP7嵌合阳性对照抗体(自制)的结合活性。
4.2生物淘选
以GPC3-huIgG1 Fc融合蛋白为目标蛋白应用生物淘选对上述鼠源免疫抗体文库进行生物淘选获得与GPC3-huIgG1 Fc融合蛋白(尤其是GPC3胞外域结合的抗体。将抗体菌种库复苏并生长至对数期后应用M13KO7辅助噬菌体挽救抗体文库，离心后用含有氨苄青霉素和卡那霉素的2xYT培养基重悬并在30℃过夜扩增。PEG/NaCl沉淀噬菌体，用甘油/PBST溶解噬菌体沉淀获得免疫库噬菌体悬液。酪蛋白封闭的噬菌体投入酪蛋白封闭的生物素化的huIgG1Fc蛋白(自制)和酪蛋白封闭的Dynabeads M-270链霉亲合素共孵育体系中，收集上清噬菌体悬液。进一步，将收集到的噬菌体悬液投入酪蛋白封闭的生物素化的GPC3-huIgG1 Fc融合蛋白 和酪蛋白封闭的Dynabeads M-270链霉亲合素共孵育体系中，用PBST清洗磁珠去除无法与GPC3-huIgG1 Fc融合蛋白结合的噬菌体。用100mM三乙胺洗脱与磁珠结合的噬菌体后用1M Tris-HCl(pH＝6.4)中和。留取10ul洗脱的噬菌体溶液用于测定输出噬菌体总量，剩余噬菌体溶液用于感染对数增长的TG1，过夜扩增后视为下一轮淘选使用的抗体文库。生物淘选共执行三轮，GPC3-huIgG1 Fc抗原浓度分别为100nM、10nM和1nM。
4.3 GPC3胞外域特异性结合克隆的筛选
将第三轮生物淘选结束后获得的抗体文库进行稀释涂布于含有氨苄青霉素的平板上获得单克隆，挑选单克隆在深孔板中过夜培养。次日利用-20℃冰箱对深孔板进行反复三次冻融，离心上清用于后续ELISA反应。ELISA反应利用山羊抗人IgG(Fab特异性的)过夜包被，依次加入离心上清，生物素化的GPC3-huIgG1 Fc融合蛋白和链霉亲合素-HRP进行阳性克隆筛选。该筛选步骤重复两次独立实验以保证数据准确。
为排除与huIgG1 Fc区结合的克隆，执行以下ELISA反应。利用山羊抗人IgG(Fab特异性的)过夜包被，依次加入离心上清，生物素化的huIgG1 Fc蛋白和链霉亲合素-HRP进行huIgG1区结合阳性克隆筛选。该筛选步骤重复两次独立实验以保证数据准确。
由以上两个步骤挑选出GPC3胞外域特异性结合克隆。
4.4利用解离速率常数koff进行亲和力排序
利用Octet K2分子互作分析仪对上述步骤筛选到GPC3胞外段特异结合克隆的冻融上清进行分析。将生物素化的GPC3-huIgG1 Fc融合蛋白固化在SA探针上，以冻融上清作为分析物，进行亲和力测定。分析步骤中只选取解离曲线计算koff数值，挑选P3H10(VH氨基酸序列如SEQ ID NO:1所示，VL氨基酸序列如SEQ ID NO:2所示)、P1C7(VH氨基酸序列如SEQ ID NO:3所示，VL氨基酸序列如SEQ ID NO:4所示)、P2G10(VH氨基酸序列如SEQ ID NO:5所示，VL氨基酸序列如SEQ ID NO:6所示)、P3A8(VH氨基酸序列如SEQ ID NO:7所示，VL氨基酸序列如SEQ ID NO:8所示)、P3G4(VH氨基酸序列如SEQ ID NO:9所示，VL氨基酸序列如SEQ ID NO:8所示)、P3D9(VH氨基酸序列如SEQ ID NO:17所示，VL氨基酸序列如SEQ ID NO:25所示)、P4F12(VH氨基酸序列如SEQ ID NO:26所示，VL氨基酸序列如SEQ ID NO:27所示)、P1F6(VH氨基酸序列如SEQ ID NO:28所示，VL氨基酸序列如SEQ ID NO:29所示)进一步开展功能研究(如表1所示)。
表1 解离速率常数测定
Sensor Location Sample Name kdis(1/s) Assoc R^2
E1 P1F6 5.94E-03 0.910044
F1 P1F6 4.94E-03 0.917047
E1 P1C7 5.28E-03 0.949309
F1 P1C7 5.34E-03 0.949602
E1 P2G10 4.29E-03 0.94705
F1 P2G10 4.67E-03 0.939041
E4 P3G4 4.09E-03 0.982496
F4 P3G4 4.59E-03 0.97781
E4 P3A8 5.03E-03 0.979674
F4 P3A8 5.13E-03 0.983841
E5 P3H10 5.95E-03 0.976471
F5 P3H10 5.72E-03 0.981531
E5 P4F12 5.71E-03 0.980821
F5 P4F12 5.50E-03 0.976893
E8 P3D9 1.98E-03 0.996878
E8 P3D9 1.85E-03 0.978959
4.5鉴定各抗体与不同物种GPC3抗原结合的种属交叉反应
利用含上述八个抗体序列噬菌粒的单克隆菌进行scFv抗体的小量原核表达并利用ESLIA鉴定scFv抗体与不同种属的GPC3的结合能力。实验独立重复两次，第二次实验由于部分样品制备失败未获取ELISA读值。
svFv抗体的原核表达具体的操作步骤如下：将单克隆菌液按照1‰的接种量接种到3mL 2YT/Amp培养基中，37℃250rpm培养过夜活化。次日，将活化的菌液按照1％接种量接种到100mL 2YT/Amp培养基中，37℃250rmp培养至OD600为0.5。向10mL 2YT/Amp菌液中加入终浓度为1mM的IPTG,并于30℃250rpm过夜诱导。过夜菌液4000rpm离心10min弃上清，将沉淀菌体重悬于2ml冰1X TES buffer(0.2M Tris-HCl(pH＝8.0),0.5mM EDTA，0.5M sucrose)中并冰浴20min。接着加入3ml的0.2X TES buffer，冰浴30min后再进行反复冻融两次，离心取上清进行超滤浓缩至300ul左右。
scFv抗体与不同种属的GPC3结合ELISA反应具体的操作步骤如下：分别包被人GPC3-his(自制)和Mouse GPC3(自制)100ng每孔于ELISA板,4℃过夜；50ul的各抗体浓缩液和50ul 1X PBST(含1％的BSA)混合后加入ELISA板中作为一抗；加入二抗为Goat anti- Mouse IgG F(ab')2Secondary Antibody(1:4000稀释，稀释液为PBST)，100ul每孔；最终显色读值。通过ELISA反应确定P3A8、P3G4、P3D9同时与人和鼠的GPC3结合均很强，ELISA结果如表2所示。
表2 种属交叉反应结果
Figure PCTCN2022101037-appb-000001
实施例3 含抗人GPC3嵌合抗原受体元件的慢病毒原液制备
1.含CAR元件的慢病毒包装主质粒制备
选取P3H10、P1C7、P2G10、P3A8、P3G4、P3D9、P4F12、P1F6克隆以及YP7克隆的抗体序列构建嵌合抗原受体。其中YP7克隆(Mitchell Ho，WO2013181543A1)是本发明的阳性对照抗体。通过分子克隆构建从N末端至C末端包含有以下结构的CAR元件慢病毒包装主质粒：CD8α信号肽，scFv抗体，CD8α铰链区，CD8α跨膜区，41BB胞质区和CD3z胞质区。挑选测序正确的克隆，接种菌液至300ml 2YT培养基中，过夜摇菌并利用质粒大提试剂盒(上海生工)完成大提质粒。
2.慢病毒包装
用阳离子聚合物PEI包装含慢病毒，流程如下：分别用无血清DMEM稀释PEI和第三代的慢病毒包装质粒(慢病毒主质粒、pMD2.G、pMDLg-pRRE、pRSV-Rev)；然后将PEI/DMEM加入质粒/DMEM混合物，涡旋震荡混匀，在室温下静置15分钟；将质粒-PEI复合物加入预先铺板的293T细胞。转染后16h换液，在48h后收集病毒上清，0.45um滤器过滤，将病毒原液以 12000g过夜离心沉淀病毒，次日弃上清用PBS溶液重悬病毒，置于负八十度冰箱储存。
实施例4 抗人GPC3嵌合抗原受体-Jurkat细胞的制备和嵌合抗原受体阳性率测定
1.病毒液的生物滴度检测
病毒液的生物滴度指每毫升中含有的具有感染能力的病毒颗粒数，准确确定病毒感染滴度有助于制备期望阳性率的嵌合抗原受体-Jurkat细胞。取24孔培养板，每孔加入梯度稀释的病毒原液，感染体积起始量为1ml，三倍倍比，五个梯度稀释，体积不足1ml，用培养基补足1ml后每孔中添加100μl含1×10 5个293T细胞的细胞悬液(含polybrene，终浓度为8μg/ml)，3天后采用流式细胞术对细胞的感染效率进行检测，使用荧光染料标记蛋白FITC-Labeled Human Glypican 3(ACRO biosystem)对细胞染色，以未感染的同步培养293T细胞作为阴性对照组(293T)。滴度计算公式为：Titer(TU/ml)＝感染细胞数×流式检测CAR表达比例/病毒液体积。生物滴度测定结果如表3所示。
表3 生物滴度测定结果
质粒编号 包装体积 慢病毒滴度 慢病毒总量
P1F6 50ml 2.02E8 TU/ml 4.00E7 TU(0.2ml)
P2G10 50ml 3.60E8 TU/ml 7.21E7 TU(0.2ml)
P1C7 50ml 1.90E8 TU/ml 3.81E7 TU(0.2ml)
P3H10 50ml 6.17E7 TU/ml 1.234E6 TU(0.2ml)
P3D9 50ml 2.99E8 TU/ml 5.97E7 TU(0.2ml)
P3A8 50ml 3.78E8 TU/ml 7.56E7 TU(0.2ml)
P3G4 50ml 3.59E8 TU/ml 7.18E7 TU(0.2ml)
P4F12 50ml 1.38E8 TU/ml 2.76E7 TU(0.2ml)
YP7 50ml 8.30E8 TU/ml 1.66E8 TU(0.2ml)
2.Jurkat细胞表型初步确定
首先通过流式细胞术检测Jurkat细胞(中科院上海药物研究所)的表型，从液氮罐中复苏并培养Jurkat细胞，收集Jurkat细胞至1.5ml离心管中，1500rpm离心5min，弃上清，管中残留约50ul液体，轻弹重悬细胞。每管加入1ml PBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。200ul PBS重悬细胞，加入1ul(PerCP)CD8抗体(Biolegend,301030)和1ul APC CD3抗体(Biolegend,300312)，室温避光10min。每管加入1mlPBS重悬细胞，1500rpm离心 5min，弃上清，轻弹重悬细胞。流式上机检测APC/PC5.5通道的阳性率。与未染色组相比，Jurkat细胞的CD3+CD8-为93.63％，所述Jurkat细胞为CD4+T淋巴细胞。结果如图2所示。
3.抗人GPC3嵌合抗原受体-Jurkat细胞的制备
从液氮中复苏并培养Jurkat细胞，取Jurkat细胞使用自动计数仪检细胞密度及活率。取1E7的细胞用于Jurkat细胞铺板和病毒感染：取1E7的细胞加入1支15ml离心管，1000rpm 3min。弃上清，用10ml 1640完全培养基进行重悬。按照每孔加入1E6/2ml/孔Jurkat细胞铺6孔细胞板。每孔加入Polybrene(1mg/mL)，使终浓度为5ug/ml加入后轻轻混匀。每孔加入MOI＝5的RD2慢病毒。加入后轻轻混匀。37℃5％CO2静置培养24h，第二天观察细胞状态并向6孔培养板中补加培养基2000ul 1640完全培养基，继续置于37℃5％CO 2培养箱静置培养。
4.抗人GPC3嵌合抗原受体-Jurkat细胞的CAR阳性率检测
病毒转导48h后进行CAR阳性率检测，使用荧光染料标记蛋白FITC-Labeled Human Glypican 3(ACRO biosystem)对细胞染色。具体流程为：收集400ul细胞至1.5ml离心管中，每管加入1000ul PBS。1500rpm离心5min，弃上清，管中残留约50ul液体，轻弹重悬细胞。每管加入1ml PBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。200ul PBS重悬细胞，每管加入配制好的的FITC-Labeled Human Glypican 3 3ul(终浓度3ug/ml)，4摄氏度避光30min。每管加入1mlPBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。300ul PBS重悬细胞上机检测。以未转导病毒的Jurkat细胞的FITC通道信号值作为阳性值域，判断各嵌合抗原受体-Jurkat细胞的阳性率。YP7-CAR Jurkat细胞阳性率为86.34％，P2G10-CAR Jurkat细胞阳性率为13.88％，P3G4-CAR Jurkat细胞阳性率为43.70％，P1F6-CAR Jurkat细胞阳性率为37.95％，P1C7-CAR Jurkat细胞阳性率为32.59％，P3H10-CAR Jurkat细胞阳性率为88.99％，P3D9-CAR Jurkat细胞阳性率为82.99％，P3A8-CAR Jurkat细胞阳性率为86.32％，P4F12-CAR Jurkat细胞阳性率为30.11％。
实施例5 基于抗人GPC嵌合抗原受体-Jurkat细胞功能的抗体初筛
1.靶细胞GPC3阳性率检测
选用GPC3阳性的HePG2细胞系作为靶细胞。首先通过流式细胞术检测HePG2细胞(中科院上海药物研究所)的表型，从液氮罐中复苏并培养HePG2细胞，收集HePG2细胞至1.5ml离心管中，1500rpm离心5min，弃上清，管中残留约50ul液体，轻弹重悬细胞。每管加入1ml PBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。200ul PBS重悬细胞，加入2ul PE Anti-Glypican 3(Abcam,ab275697)，室温避光10min。每管加入1ml PBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。流式上机检测PE通道的阳性率。与未染色组 相比，HePG2细胞的HePG2+为53.11％以上(由于分群不明显，圈门存在一定误差)。结果如图3所示。因此该HePG2细胞可以作为本研究靶细胞。
2.抗人GPC3嵌合抗原受体-Jurkat细胞激活实验
由于不同抗体克隆的CAR-jurkat细胞阳性率差异较大，因此根据GPC3-FITC测定的阳性率将细胞分为以下四组进行实验(用野生型jurkat细胞将对照YP7 CAR-jurkat细胞与各候选抗体CAR-jurkat细胞的阳性率调节一致)：
表4 根据GPC3-FITC测定的阳性率将细胞进行分组
Figure PCTCN2022101037-appb-000002
根据上表将对应的细胞加入15ml离心管，1000rpm 3min。弃上清，使用300ul的1640完全培养基继续重悬，用于各不同抗体克隆的CAR-Jurkat细胞的CD69表达的检测。
效靶细胞共孵育具体操作步骤如下：将重悬处理好靶细胞HepG2：5x10 5cells/1.25ml1640/25wells完全培养基加入96孔细胞板中(每孔加入靶细胞50uL)。将重悬处理好效应细胞Jurkat或者Jurkat 1.2E6 cells/0.3ml 1640/6 wells完全培养基按照不同效靶比加入96孔细胞 板中(E：T＝10：1每孔加入效应细胞细胞50uL；对照效应细胞组每孔加入效应细胞细胞50uL+50uL1640完全培养基。加样完成后，轻轻混匀以保证效靶细胞充分接触。将细胞置于37℃，5％CO 2培养箱中培养过夜。
Jurkat细胞表面CD69表达检测详细操作步骤如下：收集细胞至1.5ml离心管中，每管加入1000ul PBS。1500rpm离心5min，弃上清，轻弹重悬细胞。每管加入1mlPBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。每管加入200ul PBS重悬细胞后，加入1ul APC抗人CD69抗体，室温避光染色10min。每管加入1mlPBS重悬细胞，1500rpm离心5min，弃上清，轻弹重悬细胞。300ul PBS重悬细胞后上机检测。根据APC通道数值评估CD69表达水平。根据上述Mean APC作图，三组实验的结果分别如图4，图5和图6。
通过以上数据确定，只有P3D9的效靶比10:1样品的平均APC荧光强度与对照样品(无靶细胞)平均APC荧光强度的比值略高于YP7组，可以判断P3D9具更优的激活T细胞的能力。
进一步的，利用上述同样的方法再次制备了一批YP7 CAR-jurkat细胞(阳性率70.48％)和P3D9 CAR-jurkat(阳性率52.72％)。用野生型jurkat细胞将对照YP7 CAR-jurkat细胞的阳性率调节为52.72％，以效靶比10:1条件完成效靶细胞共孵育，两组Jurkat细胞表面CD69表达情况如图7所示。结果显示P3D9的效靶比10:1样品的平均APC荧光强度与对照样品(无靶细胞)平均APC荧光强度的比值高于YP7组，因此P3D9具更优的激活T细胞的能力。
实施例6 基于抗人GPC嵌合抗原受体-T细胞功能的抗体验证
1.抗人GPC3嵌合抗原受体-T细胞制备
用来自于正常供体的T细胞(妙顺生物)以及实施例3.2中所述的含P3H10、P3D9和YP7抗体序列的慢病毒分别制备P3H10-CAR-T细胞、P3D9-CAR-T细胞和YP7-CAR-T细胞。
2.HepG2-luc-GFP靶细胞的构建
首先制备靶细胞构建所需的慢病毒：通过化学合成的方式，合成以内部核糖体进入位点(IRES)串联的萤光素酶和绿色荧光蛋白基因。通过分子克隆构建慢病毒包装主质粒(PCCL-LUC-GFP)并包装慢病毒。用上述慢病毒分别转染实施例5.1所述的HepG2细胞后，以绿色荧光蛋白作为标记筛选出绿色荧光蛋白高表达的HepG2-luc-GFP单细胞克隆，扩大培养作为CAR-T杀伤实验靶细胞。
3.抗人GPC3嵌合抗原受体-T细胞杀伤靶细胞
CAR-T杀伤实验通过检测CAR-T细胞体外对靶细胞的杀伤效果来评估CAR-T细胞的体外功能。以不同效靶比(以3E4个靶细胞为基准，效应细胞分别为两倍、五倍及十倍的靶细 胞数)将T细胞与HepG2-LUC-GFP靶细胞共同培养，同时设置靶细胞和未转染CAR元件T细胞混合的阴性对照组(NT)。实验设置最大释放量孔，实验结束时通过反复冻融裂解细胞，取细胞上清检测荧光值即为最大释放量荧光值。18小时后在培养体系中加入萤光素酶反应底物，检测荧光值，通过以下公式计算杀伤效率：杀伤效率＝实验孔荧光值/最大释放量孔荧光值x100％。实验分组及结果见图8。实验数据显示，在各效靶比下，P3D9-CAR-T细胞对靶细胞的杀伤率均高于阳性对照YP7-CAR-T细胞，而P3H10-CAR-T细胞在效靶比为5:1和10:1时杀伤率均低于阳性对照YP7-CAR-T细胞。
实施例7 GPC3×CD3双特异抗体对T细胞信号的激活能力检测
1.双特异性抗体制备
将下表所述抗体序列构建于pTT5真核表达载体，转染HEK293细胞，表达6天后收集上清通过protein A亲和层析纯化。SEC-HPLC检测显示两个抗体的单体含量均约为90％，可用于对T细胞信号激活能力的检测。
表5 双特异性抗体序列
双特异性抗体名称 序列编号
实验系统对照双抗(PSMA×CD3) SEQ ID NO:47
P3D9双抗(GPC3×CD3) SEQ ID NO:48
YP7双抗(GPC3×CD3) SEQ ID NO:49
2.PSMA×CD3双特异抗体靶细胞构建
通过化学合成的方式合成人前列腺特异性膜抗原(PSMA)(UniProtKB/Swiss-Prot accession No.Q04609)的第1位甲硫氨酸至第750位丙氨酸区间的基因序列。通过分子克隆，将PSMA基因片段扩增产物用TaKaRa无缝克隆试剂盒克隆到慢病毒包装主质粒(PCCL-LUC-GFP)并包装慢病毒，转导HEK293T细胞。以GFP荧光标记通过流式分选仪(SONY SH800)挑选单克隆细胞系作为PSMA×CD3双特异抗体靶细胞。
3.构建jurkat-NFTA luc报告系统稳转细胞系
将pGL4.3[luc2P/NFAT-RE](Promega)质粒转染Jurkat细胞后用200ug/ml潮霉素加压筛选，长期培养后通过流式分选仪(SONY SH800)挑选单克隆细胞系作为Jurkat-NFAT luc报告细胞。
4.T细胞信号刺激检测
双特异性抗体活性的定性分析拟采用终浓度为1E4pM、1E3pM、0pM三个浓度梯度进 行试验，将1640完全培养基预先配置为5E4pM和5E3pM两个浓度。将靶细胞重悬为1E6个细胞/ml，按每孔40ul体积加入96孔板中，再将Jurkat-NFAT luc重悬为1E6个细胞/ml，按每孔40ul体积加入已有靶细胞的96孔板中，将各梯度含抗体的培养基按每孔20ul体积加入96孔板中(0pM组加入培养基)，混合共孵育五小时。
从培养箱中取出待测细胞培养板，室温放置20min左右，使培养板温度平衡至室温。每孔加入100μL平衡至室温的One-Lite TM检测试剂，等待约5min使细胞充分裂解，转移180ul到96孔白板中，酶标仪进行读数。选择酶标仪的发光检测模块，在加入检测试剂后的15min内完成读板。实验系统对照双抗组的荧光信号值随着抗体浓度的提高而显著提高表明jurkat T细胞信号受到了基于双抗的靶向刺激，在P3D9双抗组中荧光信号随着抗体浓度提高而升高证明该双抗能有效刺激Jurkat T细胞，在YP7双抗组中各浓度梯度均未观测到荧光信号的升高表明该抗体结构在所述浓度时无法有效刺激Jurkat T细胞。该实验通过定性分析确定P3D9双抗具生物学活性，实验结果详见下表：
表6 T细胞信号刺激检测结果
Figure PCTCN2022101037-appb-000003

Claims (70)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:17所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
  6. 根据权利要求4-5中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:13所示的氨基酸序列。
  7. 根据权利要求4-6中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:14所示的氨基酸序列。
  8. 根据权利要求3-7中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:15所示的氨基酸序列。
  9. 根据权利要求2-8中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:16所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:13所示的氨基酸序列，所述H-FR2包含SEQ ID NO:14所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:16所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含VH，所述VH包含SEQ ID NO:17所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定 区。
  14. 根据权利要求12-13中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG1恒定区。
  15. 根据权利要求1-14中任一项所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:25所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含LCDR3，所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含LCDR2，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含LCDR1，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
  20. 根据权利要求18-19中任一项所述的分离的抗原结合蛋白，其包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:21所示的氨基酸序列。
  21. 根据权利要求18-20中任一项所述的分离的抗原结合蛋白，其包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2包含SEQ ID NO:22所示的氨基酸序列。
  22. 根据权利要求17-21中任一项所述的分离的抗原结合蛋白，其包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3包含SEQ ID NO:23所示的氨基酸序列。
  23. 根据权利要求16-22中任一项所述的分离的抗原结合蛋白，其包含L-FR4，所述L-FR4的N端与所述LCDR3的C端直接或间接相连，且所述L-FR4包含SEQ ID NO:24所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:21所示的氨基酸序列，所述L-FR2包含SEQ ID NO:22所示的氨基酸序列，所述L-FR3包含SEQ ID NO:23所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:24所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的分离的抗原结合蛋白，其包含VL，所述VL包含 SEQ ID NO:25所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其包含抗体轻链恒定区。
  27. 根据权利要求26所述的分离的抗原结合蛋白，其中所述轻链恒定区源自人Igκ恒定区。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包含VH和VL，所述VH包含SEQ ID NO:17所示的氨基酸序列，且所述VL包含SEQ ID NO:25所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的分离的抗原结合蛋白，其包含抗体或其抗原结合片段。
  30. 根据权利要求29所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
  31. 根据权利要求29-30中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其能够特异性结合磷脂酰肌醇蛋白多糖3(GPC3)。
  33. 根据权利要求32所述的分离的抗原结合蛋白，其中所述GPC3包含人GPC3和/或鼠GPC3。
  34. 嵌合抗原受体，其包含抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述抗原结合结构域包含权利要求1-33中任一项所述的分离的抗原结合蛋白。
  35. 根据权利要求34所述的嵌合抗原受体，其中所述抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
  36. 根据权利要求34-35中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:18所示的氨基酸序列。
  37. 根据权利要求34-36中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO:17所示的氨基酸序列。
  38. 根据权利要求34-37中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO:25所示的氨基酸序列。
  39. 根据权利要求34-38中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含SEQ  ID NO:34所示的氨基酸序列。
  40. 根据权利要求34-39中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含抗体或其抗原结合片段。
  41. 根据权利要求34-40中任一项所述的嵌合抗原受体，其中所述抗原结合结构域包含scFv。
  42. 根据权利要求34-41中任一项所述的嵌合抗原受体，其中所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  43. 根据权利要求42所述的嵌合抗原受体，其中所述跨膜结构域为CD8α跨膜区。
  44. 根据权利要求34-43中任一项所述的嵌合抗原受体，其中所述跨膜结构域包含SEQ ID NO:36所示的氨基酸序列。
  45. 根据权利要求34-44中任一项所述的嵌合抗原受体，其中所述共刺激域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
  46. 根据权利要求45所述的嵌合抗原受体，其中所述共刺激域为4-1BB胞质区。
  47. 根据权利要求34-46中任一项所述的嵌合抗原受体，其中所述共刺激域包含SEQ ID NO:37所示的氨基酸序列。
  48. 根据权利要求34-47中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
  49. 根据权利要求34-48中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包含SEQ ID NO:38所示的氨基酸序列。
  50. 根据权利要求34-49中任一项所述的嵌合抗原受体，其还包含铰链区，所述铰链区包含CD8α铰链区。
  51. 根据权利要求50所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:35所示的氨基酸序列。
  52. 根据权利要求34-51中任一项所述的嵌合抗原受体，其还连接信号肽。
  53. 根据权利要求52所述的嵌合受体，其中所述信号肽包含CD8α信号肽。
  54. 根据权利要求34-53中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:33所示的氨基酸序列。
  55. 根据权利要求34-54中任一项所述的嵌合抗原受体，其包含SEQ ID NO:39所示的氨基酸序列。
  56. 免疫效应细胞，其包含权利要求34-55中任一项所述的嵌合抗原受体。
  57. 根据权利要求56所述的免疫效应细胞，其中所述免疫效应细胞包含T细胞。
  58. 多特异性抗原结合蛋白，其包含权利要求1-33中任一项所述的分离的抗原结合蛋白。
  59. 根据权利要求58所述的多特异性抗原结合蛋白，其包含靶向CD3的部分。
  60. 根据权利要求59所述的多特异性抗原结合蛋白，其中所述CD3包含人CD3。
  61. 分离的核酸分子，其编码权利要求1-33中任一项所述的分离的抗原结合蛋白、权利要求34-55中任一项所述的嵌合抗原受体或权利要求58-60中任一项所述的多特异性抗原结合蛋白。
  62. 载体，其包含权利要求61所述的核酸分子。
  63. 宿主细胞，其包含权利要求61所述的核酸分子或权利要求62所述的载体。
  64. 药物组合物，其包含权利要求1-33中任一项所述的抗原结合蛋白，权利要求34-55中任一项所述嵌合抗原受体、权利要求56-57中任一项所述的免疫效应细胞或权利要求58-60中任一项所述的多特异性抗原结合蛋白，以及任选地药学上可接受的载剂。
  65. 制备权利要求1-33中任一项所述的分离的抗原结合蛋白、权利要求34-55中任一项所述的嵌合抗原受体、权利要求56-57中任一项所述的免疫效应细胞、权利要求58-60中任一项所述的多特异性抗原结合蛋白或权利要求64所述的药物组合物的方法。
  66. 权利要求1-33中任一项所述的分离的抗原结合蛋白、权利要求34-55中任一项所述的嵌合抗原受体、权利要求56-57中任一项所述的免疫效应细胞、权利要求58-60中任一项所述的双特异性抗原结合蛋白或权利要求64所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  67. 根据权利要求66所述的用途，其中所述疾病和/或病症包含GPC3相关疾病和/或病症。
  68. 根据权利要求66-67中任一项所述的用途，其中所述疾病和/或病症包含肿瘤。
  69. 根据权利要求68所述的用途，其中所述肿瘤包括实体瘤和/或血液瘤。
  70. 检测样品中GPC3存在和/或含量的试剂盒，所述试剂盒包括权利要求1-33中任一项所述的分离的抗原结合蛋白、权利要求34-55中任一项所述的嵌合抗原受体、权利要求56-57中任一项所述的免疫效应细胞、权利要求58-60中任一项所述的多特异性抗原结合蛋白、权利要求61所述的核酸分子、权利要求62所述的载体、权利要求63所述的宿主细胞和/或权利要求64所述的药物组合物。
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