WO2023001155A1 - 一种磷脂酰肌醇蛋白聚糖3抗体及其应用 - Google Patents

一种磷脂酰肌醇蛋白聚糖3抗体及其应用 Download PDF

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WO2023001155A1
WO2023001155A1 PCT/CN2022/106547 CN2022106547W WO2023001155A1 WO 2023001155 A1 WO2023001155 A1 WO 2023001155A1 CN 2022106547 W CN2022106547 W CN 2022106547W WO 2023001155 A1 WO2023001155 A1 WO 2023001155A1
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antibody
glypican
seq
amino acid
light chain
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PCT/CN2022/106547
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French (fr)
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张文军
刘敏
田文武
李峰
覃陆英
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广州爱思迈生物医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the disclosure belongs to the technical fields of tumor treatment and molecular immunology, and relates to a Glypican 3 antibody and applications thereof.
  • Hepatocellular carcinoma hepatocellular carcinoma, HCC
  • HCC Hepatocellular carcinoma
  • Glypican-3 is a specific membrane protein on the cell surface of hepatocellular carcinoma (HCC) discovered in recent years, and it is a good target for targeted therapy of HCC.
  • GPC3 is a heparan sulfate (HS) glycoprotein bound to the surface of the cell membrane. Its basic structure is composed of core protein, heparan sulfate chain and glycosylated phosphatidylinositol. The size is about 66kD.
  • Glycosylated phosphatidylinositol (GPI) is covalently bound to the cell membrane, and the amino terminal is free outside the cell. There are multiple disulfide bonds inside it to make it have a spherical three-dimensional structure.
  • GPC3 protein can be found in 358 Arginine at position 359 and arginine at position 359 are cleaved by Furin protease and divided into two subunits, forming a 30kD C-terminal subunit and a 40kD free N-terminal subunit, wherein the C-terminal subunit
  • the unit can be combined with two heparan sulfate chains (HS chains), and remain in the membrane and cytoplasm of tumor cells; the N-terminal subunit enters the circulatory system and becomes soluble GPC3 molecules secreted into the blood.
  • HS chains heparan sulfate chains
  • the HS group of GPC3 can interact with growth factors and their receptors, extracellular matrix proteins, and adhesion molecules, and participate in the regulation of cell proliferation, differentiation, adhesion, and migration.
  • the secreted way activates the canonical Wnt signaling pathway to make the Wnt gene highly expressed, and the expression products of the Wnt gene can promote the growth of various tumor tissues.
  • GPC3 expression is characterized by overexpression in liver cancer tissues, but no or low expression in normal human tissues. Based on the unique structural and functional characteristics of GPC3, using it as a therapeutic target for liver cancer may become a tool for the treatment of liver cancer. sword.
  • the present disclosure provides a Glypican 3 antibody, the heavy chain complementarity determining region of the Glypican 3 antibody includes the amino acid sequence shown in SEQ ID No. 6-8;
  • the light chain complementarity determining region of the Glypican 3 antibody includes the amino acid sequences shown in SEQ ID No. 14-16.
  • the heavy chain variable region of the Glypican 3 antibody includes the amino acid sequence shown in SEQ ID No.1; the light chain of the Glypican 3 antibody can be The variable region includes the amino acid sequence shown in SEQ ID No.9.
  • the present disclosure also provides an affinity matured Glypican 3 antibody, the affinity matured Glypican 3 antibody is prepared from the aforementioned Glypican 3 antibody through mutation treatment.
  • the heavy chain complementarity determining region of the affinity matured Glypican 3 antibody includes the amino acid sequences shown in SEQ ID No. 6-8.
  • the light chain complementarity determining region CDR1 of the affinity matured Glypican 3 antibody includes the amino acid sequence shown in SEQ ID No.14, and the light chain complementarity determining region CDR2 includes SEQ ID No.15
  • the light chain complementarity determining region CDR3 includes any one of the amino acid sequences shown in SEQ ID No.20-40.
  • the light chain complementarity determining region CDR1 of the affinity matured Glypican 3 antibody includes the amino acid sequence shown in any one of SEQ ID No. 17-19, and the light chain complementarity determining region CDR2 Including the amino acid sequence shown in SEQ ID No.15, the light chain complementarity determining region CDR3 includes the amino acid sequence shown in SEQ ID No.16.
  • the heavy chain framework region of the affinity matured Glypican 3 antibody comprises the amino acid sequence shown in SEQ ID No.2-5; the light chain framework region of the antibody comprises SEQ ID Amino acid sequences shown in No.10-13.
  • the present disclosure also provides a biological material that is any of the following B1) to B6):
  • B1) A nucleic acid molecule encoding the heavy chain and/or light chain in the antibody of (b1) or (b2) or the antigen-binding portion of the antibody of (b1) or (b2): (b1) the phospholipid described above Glypican 3 antibody, (b2) the affinity matured Glypican 3 antibody of any one of the above;
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);
  • B5 the cell line containing the nucleic acid molecule described in B1), or the cell line containing the expression cassette described in B2), or the cell line containing the recombinant vector described in B3);
  • the present disclosure also provides a pharmaceutical composition, which includes the Glypican 3 antibody and/or the affinity-matured Glypican 3 antibody described above.
  • the pharmaceutical composition further comprises one or more therapeutic agents.
  • the therapeutic agent comprises at least one of the following: a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent for radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent.
  • the present disclosure also provides the application of the Glypican 3 antibody, the affinity-matured Glypican 3 antibody described above, or the pharmaceutical composition in the preparation of a drug for treating tumors .
  • the tumor comprises liver cancer.
  • the present disclosure also provides the use of the Glypican 3 antibody, the affinity-matured Glypican 3 antibody described above, or the pharmaceutical composition for treating tumors.
  • the tumor comprises liver cancer.
  • the present disclosure also provides a method for treating tumors, the method comprising: administering the Glypican 3 antibody, the affinity maturation method described in any one of the above to the subject in need thereof. Glypican 3 antibody or the pharmaceutical composition.
  • the tumor comprises liver cancer.
  • Figure 1A is a diagram of the binding activity of the 10C7 antibody to the antigen in the extracellular region of GPC3;
  • Figure 1B is a diagram of the binding activity of the 10C7 antibody to the GPC3 proximal membrane antigen
  • Figure 2A is a diagram of the binding activity of 10C7 antibody to 293F/GPC3 full-length cells
  • Figure 2B is a diagram of the binding activity of 10C7 antibody to 293F/GPC3 near-membrane cells
  • Figure 3A is a diagram of the binding activity of the affinity matured variants (5D1, 8G2, 5H9 and 8H5) to the GPC3 proximal membrane antigen;
  • Figure 3B is a diagram of the binding activity of the affinity matured variants (5C1, 7F11 and 5H1) to the GPC3 proximal membrane antigen;
  • Figure 3C is a diagram of the binding activity of the affinity matured variants (7H3, 11C6, 7A2 and 1A6) to the GPC3 proximal membrane antigen;
  • Figure 3D is a diagram of the binding activity of the affinity matured variants (5A5, 8F1, 5E11 and 5B6) to the GPC3 proximal membrane antigen;
  • Figure 3E is a diagram of the binding activity of the affinity matured variants (5A4, 5D8, 5B10 and 5B8) to the GPC3 proximal membrane antigen;
  • Figure 3F is a diagram of the binding activity of the affinity matured variants (7C6, 8F9, 9G4 and 7F1) to the GPC3 proximal membrane antigen;
  • Figure 3G is a diagram of the binding activity of the affinity matured variant 7A7 to the GPC3 proximal membrane antigen
  • Figure 4A is a graph of the binding activity of antibodies (hIgG, GC33 monoclonal antibody mimics, 5D1 and 8G2) detected by FACS to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 4B is a diagram of the binding activity of FACS detection antibodies (5H9, 8H5, 5C1 and 7F11) to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 4C is a diagram of the binding activity of FACS detection antibodies (5H1, 7H3, 11C6 and 7A2) to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 4D is a diagram of the binding activity of antibodies (1A6, 5A5, 8F1 and 5E11) detected by FACS to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 4E is a diagram of the binding activity of FACS detection antibodies (5B6, 5A4, 5D8 and 5B10) to cells overexpressing human GPC3 proximal membrane antigen;
  • Fig. 4F is a diagram of the binding activity of FACS detection antibodies (5B8, 7C6, 8F9 and 9G4) to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 4G is a graph showing the binding activity of antibodies (7F1, 7A7) detected by FACS to cells overexpressing human GPC3 proximal membrane antigen;
  • Figure 5A is a diagram of the binding activity of antibodies (hIgG, GC33 monoclonal antibody mimics, 5D1 and 8G2) detected by FACS to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5B is a diagram of the binding activity of FACS detection antibodies (5H9, 8H5, 5C1 and 7F11) to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5C is a graph of the binding activity of antibodies (5H1, 7H3, 11C6 and 7A2) detected by FACS to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5D is a diagram of the binding activity of FACS detection antibodies (1A6, 5A5, 8F1 and 5E11) to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5E is a diagram of the binding activity of FACS detection antibodies (5B6, 5A4, 5D8 and 5B10) to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5F is a diagram of the binding activity of FACS detection antibodies (5B8, 7C6, 8F9 and 9G4) to cells overexpressing the full-length antigen of human GPC3;
  • Figure 5G is a graph showing the binding activity of antibodies (7F1, 7A7) detected by FACS to cells overexpressing the full-length antigen of human GPC3;
  • Figure 6A is a graph showing the detection results of GPC3 ⁇ CD3 bispecific antibodies (5H9 ⁇ CD3, 10C7 ⁇ CD3, 5D1 ⁇ CD3 and hIgG) on T cell activation;
  • Figure 6B is a graph showing the detection results of GPC3 ⁇ CD3 bispecific antibodies (7C6 ⁇ CD3, 7A7 ⁇ CD3, 8F9 ⁇ CD3, 7F11 ⁇ CD3 and 7F1 ⁇ CD3) on T cell activation;
  • Fig. 7A is a diagram showing the detection results of the killing effect of GPC3 ⁇ CD3 bispecific antibodies (7C6 ⁇ CD3, 7F11 ⁇ CD3, 7F1 ⁇ CD3, 7A7 ⁇ CD3, 8F9 ⁇ CD3, 5D1 ⁇ CD3 and 5H9 ⁇ CD3) on HepG2 cells;
  • Figure 7B is a graph showing the killing effect detection results of GPC3 ⁇ CD3 bispecific antibodies (7C6 ⁇ CD3, 7F11 ⁇ CD3, 7F1 ⁇ CD3, 7A7 ⁇ CD3, 8F9 ⁇ CD3, 5D1 ⁇ CD3 and 5H9 ⁇ CD3) on Huh-7 cells ;
  • Fig. 8 is a graph showing the volume change of subcutaneous CT26-hGPC3 tumors in BALB/c-hCD3E mice.
  • Antibody antibody, Ab.
  • Immunoglobulin immunoglobulin, Ig.
  • Heavy chain heavy chain, HC.
  • Light chain light chain, LC.
  • Heavy chain variable domain heavy chain variable domain, VH.
  • Heavy chain constant domain heavy chain constant domain, CH.
  • Light chain variable domain light chain variable domain, VL.
  • Light chain constant domain light chain constant domain, CL.
  • Antigen binding region antigen binding fragment, Fab.
  • Hinge area hinge region.
  • Fc fragment fragment crystallizable region, Fc region.
  • Monoclonal antibodies monoclonal antibodies, mAbs.
  • Antibody-dependent cytotoxicity antibody-dependent cell-mediated cytotoxicity, ADCC.
  • Complement-dependent cytotoxicity complement dependent cytotoxicity, CDC.
  • Natural killer cells natural killing cells, NK cells.
  • Bispecific antibody bispecific antibody, BsAb.
  • T cell receptor T cell receptor, TCR.
  • Major histocompatibility complex major histocompatibility complex, MHC.
  • Complementarity determining region refers to the antigen complementary binding region of an antibody.
  • Immunoreceptor tyrosine-based activation motif immunoreceptor tyrosine-based activation motif, ITAM.
  • Single-chain variable region antibody fragment also known as single-chain antibody: single-chain variable fragment, scFv;
  • Adoptive immunotherapy adoptive cellular immunotherapy, ACI.
  • Lymphokine-activated killer cells lymphokine-activated killer cells, LAK cells.
  • Tumor infiltrating lymphocytes Tumor Infiltrating Lymphocyte, TIL cells.
  • Cytokine-induced killer cells cytokine-induced killer cell, CIK cells.
  • nucleotide sequences described in this disclosure are arranged and written left to right in a 5' to 3' orientation.
  • amino acid sequences described in this disclosure are arranged and written left to right in a direction from amino terminus (N-terminus) to carboxy-terminus (C-terminus).
  • amino acid refers to one of the 20 naturally occurring amino acids or any unnatural analogue that may be present at a specific defined position.
  • amino acid mutation in the present disclosure refers to amino acid substitution, insertion, deletion and modification in the polypeptide sequence, and any combination of amino acid substitution, insertion, deletion and modification. Alternative amino acid modifications herein are substitutions.
  • amino acid substitution or “substitution” in the present disclosure refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid.
  • a substitution C220S refers to a variant polypeptide in which the aminocysteine at position 220 of the polypeptide has been replaced by the amino acid serine.
  • Amino acid mutations can be achieved by molecular cloning or chemical methods. Molecular cloning methods include PCR, site-directed mutagenesis, and total gene synthesis.
  • protein refers to molecules with two or more amino acids linked by peptide bonds, including natural proteins, artificial proteins, protein fragments, mutant proteins and fusion proteins.
  • structural domain refers to a specific structural region with independent functions in a biomacromolecule.
  • the domain has an independent tertiary structure, and its function does not depend on the rest of the biomacromolecule.
  • the domain in this disclosure refers specifically to a protein In such regions, such as the VH domain of the heavy chain variable region and the VL domain of the light chain variable region, the combination of domains can form a large domain.
  • antibody refers to an immunoglobulin molecule that contains at least one antigen recognition site and is capable of specifically binding an antigen.
  • antigen is a substance that can induce an immune response in the body and specifically binds to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the above substances .
  • the binding of antibody and antigen is mediated by the interaction formed between them, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the region on the surface of an antigen that binds to an antibody is called an "antigenic determinant" or "epitope".
  • each antigen has multiple determinants.
  • antibody referred to in this disclosure includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies comprising at least two different epitope binding domains (eg, bispecific antibodies), human antibodies, humanized antibodies, post-translationally modified antibodies, camelid antibodies, chimeric antibodies, fusion proteins comprising antibody epitopes, and any other modified immunoglobulin molecule comprising an antigen recognition site, provided These antibodies exhibit the desired biological activity.
  • Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen binding site.
  • Fab fragment
  • Fab region fragment
  • VH variable domain
  • VL variable domain
  • Fc is the effector region of an antibody, capable of eliciting, for example, CDC, ADCC, ADCP, cytokine release, and the like.
  • Natural antibody Fc is usually composed of two identical protein fragments, which contain two or three immunoglobulin constant region domains.
  • the Fc described in the present disclosure includes native Fc as well as mutated Fc. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined to comprise residues starting at C226 or P230 to its carboxyl terminus. Under the experimental conditions, the fragments generated by papain digestion of immunoglobulin monomers are Fab and Fc, respectively.
  • the "hinge” or “hinge region” of an antibody refers to a flexible polypeptide comprising the amino acids between the first and second constant domains (CH1 and CH2) of the antibody.
  • the amino acid numbering of antibody variable regions described in the present disclosure uses the coding scheme described by Kabat et al. in 1991, namely "Kabat index” or “Kabat numbering” (Kabat, E.A. et al. Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.: 1991).
  • the amino acid numbering of the constant regions of antibodies described in this disclosure uses the EU index (Edelman GM, et. al. Proc Natl Acad Sci US A 1969, 63:78-85.).
  • antigen-binding site refers to one or more amino acid residues that directly interact with an antigen in an antigen-binding molecule.
  • the antigen-binding site of an antibody is composed of complementarity-determining regions (CDRs).
  • CDRs complementarity-determining regions
  • Natural immunoglobulin molecules usually contain two Fab molecules usually contain one antigen-binding site.
  • T cell activation refers to one or more immune responses of T lymphocytes, especially killer T lymphocytes, including: proliferation, differentiation, release of cytokines, secretion of killer effector molecules, cell killing, etc.
  • EC 50 namely the concentration for 50% of maximal effect, refers to the concentration of antibody corresponding to causing 50% of the maximal effect.
  • binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen to which it is directed.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10-5M, such as less than about 10-6M, 10-7M, 10-8M , 10-9M, or 10-10M or less binding affinity (KD) to the antigen.
  • KD binding affinity
  • targeting refers to specific binding.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which describes the binding affinity between an antibody and an antigen.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10-5M, eg, less than about 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M or less.
  • single-chain variable region antibody fragment refers to a fusion protein of an immunoglobulin heavy chain variable region VH and a light chain variable region VL, including different combinations of N-terminal VH and N-terminal VL, It can be prepared by conventional molecular cloning methods for constructing recombinant proteins (Sambrook JF, E.F. et al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing part or all of the CDR regions of a human immunoglobulin (recipient antibody) with a CDR region of a non-human antibody (donor antibody), wherein
  • the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody of desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize one of the antibodies. or multiple properties.
  • the term "host cell” refers to a cell into which an exogenous nucleic acid has been introduced, and its progeny can be transformed or transfected with a nucleotide encoding the polypeptide, thereby expressing the exogenous polypeptide.
  • the host cells described in the present disclosure include but are not limited to CHO cells (Chinese hamster ovary cells, Chinese hamster ovary cells), HEK293 cells (Human embryonic kidney cells 293, human embryonic kidney cells 293), BHK cells (Baby Hamster Kidney, young hamster cells) kidney cells), myeloma cells, yeast, insect cells or prokaryotic cells such as Escherichia coli, etc.
  • the "host cell” mentioned in the present disclosure not only refers to the cell into which the exogenous nucleic acid has been introduced, but also includes the offspring of the cell. Since the offspring cells will undergo mutations during cell division, they still belong to the host cell described in the present disclosure. Term scope.
  • the present disclosure further encompasses nucleic acid sequences encoding these polypeptide chains.
  • the nucleic acid sequence is inserted into a suitable vector, including but not limited to: plasmid, phage expression vector, cosmid, artificial chromosome, phage and animal virus.
  • the expression vector contains elements for regulating expression, including but not limited to promoter, transcription initiation sequence, enhancer, signal peptide sequence and the like. Promoters include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, ⁇ -actin promoter, EF-1 ⁇ promoter, CMV promoter, and SV40 promoter.
  • Appropriate methods known in the art can be used to transfer the expression vector into host cells, including but not limited to: calcium phosphate precipitation method, polyethylenimine transfection method, liposome transfection method, electroporation method, PEI ( Polyethyleneimine) transfection method.
  • an "effective amount” may refer to an amount of a composition described herein or a pharmaceutical formulation described herein that will cause the desired effect in a tissue, system, animal, plant, plant, or animal, as the researcher, veterinarian, physician, or other clinician is seeking. Desired biological or medical response of protozoa, bacteria, yeast or humans.
  • the term "subject” refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • Some embodiments of the present disclosure provide a Glypican 3 antibody and applications thereof, the Glypican 3 antibody can specifically bind to Glypican-3 with high affinity, It can activate T lymphocytes, and has important application prospects in improving immune cell activity and enhancing immune response, preventing and treating tumors and infectious diseases.
  • Some embodiments of the present disclosure provide a Glypican 3 antibody, the heavy chain complementarity determining regions (VH-CDR1, VH-CDR2 and VH-CDR3) of the Glypican 3 antibody comprising The amino acid sequence shown in SEQ ID No.6-8, the light chain complementarity determining region (VL-CDR1, VL-CDR2 and VL-CDR3) of described glypican 3 antibody comprises SEQ ID No.14- The amino acid sequence shown in 16.
  • the Glypican-3 antibody can specifically bind to Glypican-3 with high affinity, can activate T lymphocytes, and can improve immune cell activity and enhance immune response, prevent It has important application prospects in the treatment of tumors and infectious diseases.
  • the Glypican 3 antibody has the following properties:
  • the heavy chain variable region of the Glypican 3 antibody comprises the amino acid sequence shown in SEQ ID No. 1, and the light chain of the Glypican 3 antibody can be The variable region includes the amino acid sequence shown in SEQ ID No.9.
  • the heavy chain framework region of the Glypican 3 antibody comprises the amino acid sequence shown in SEQ ID No. 2-5, and the light chain of the Glypican 3 antibody The chain framework region includes the amino acid sequences shown in SEQ ID No. 10-13.
  • SEQ ID No. 2 QVQLVQSGAEVKKPGASVKLSCKAS.
  • SEQ ID No. 3 MHWVKQTPGKGLKWIGA.
  • SEQ ID No. 4 AYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYC.
  • SEQ ID No.6 (VH-CDR1): GYTFTDYE.
  • SEQ ID No. 7 (VH-CDR2): LDPKTGDT.
  • SEQ ID No. 8 (VH-CDR3): TRFYSYTY.
  • SEQ ID No. 10 DIVLTQSPLSLTVTPGEPASISCRSS.
  • SEQ ID No. 11 LSWLQQRPGQPPRLLIY.
  • SEQ ID No. 12 NRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC.
  • SEQ ID No. 13 FGQGTKVEIK.
  • SEQ ID No. 14 (VL-CDR1): ESLVHSNGNTY.
  • SEQ ID No. 16 (VL-CDR3): TQATQFPYT.
  • Some embodiments of the present disclosure also provide a DNA fragment encoding the above-mentioned Glypican 3 antibody.
  • the light chain of the Glypican 3 antibody has the nucleotide sequence shown in SEQ ID NO.41, and the heavy chain of the Glypican 3 antibody has SEQ ID NO. The nucleotide sequence of ID NO.42.
  • Some embodiments of the present disclosure also provide an expression vector comprising at least one copy of the above-mentioned DNA fragment of the Glypican 3 antibody.
  • Some embodiments of the present disclosure also provide a host cell comprising the above-mentioned expression vector.
  • Some embodiments of the present disclosure also provide an affinity matured Glypican 3 antibody, wherein the affinity matured Glypican 3 antibody is mutated from the Glypican 3 antibody described above Prepared by processing.
  • the mutagenesis comprises random mutation of the light chain variable region and/or the heavy chain variable region of the Glypican 3 antibody.
  • the mutations obtained by random mutation of the light chain variable region and/or heavy chain variable region of the Glypican 3 antibody are as described in Table 2. It is believed, without being bound by theory, that the random mutations further increase the affinity of the glypican 3 antibody.
  • the heavy chain complementarity determining region of the affinity matured Glypican 3 antibody includes the amino acid sequences shown in SEQ ID No. 6-8.
  • the light chain complementarity determining region CDR1 of the affinity matured Glypican 3 antibody includes the amino acid sequence shown in SEQ ID No.14, and the light chain complementarity determining region CDR2 includes SEQ ID No.15
  • the light chain complementarity determining region CDR3 includes any one of the amino acid sequences shown in SEQ ID No.20-40.
  • the light chain complementarity determining region CDR1 of the affinity matured Glypican 3 antibody includes the amino acid sequence shown in any one of SEQ ID No. 17-19, and the light chain complementarity determining region CDR2 Including the amino acid sequence shown in SEQ ID No.15, the light chain complementarity determining region CDR3 includes the amino acid sequence shown in SEQ ID No.16.
  • SEQ ID No. 17 HPLLHSNGNTY.
  • SEQ ID No. 18 ESLVHINGNTY.
  • SEQ ID No. 19 QSLLSSNGNTY.
  • SEQ ID No. 20 TQSAFAPYT.
  • SEQ ID No. 21 VQVSFFPYT.
  • SEQ ID No. 22 TQLTRVPYT.
  • SEQ ID No. 23 FQNGRFPYT.
  • SEQ ID No. 24 TQATRPPYS.
  • SEQ ID No. 25 VQVSSFPYT.
  • SEQ ID No. 26 TQATHLYYS.
  • SEQ ID No. 27 TQATRPPYL.
  • SEQ ID No. 28 VQVSAFPYT.
  • SEQ ID No. 29 LQVSIFPYT.
  • SEQ ID No. 30 VQVSRFPYT.
  • SEQ ID No. 31 VQTATFPYT.
  • SEQ ID No. 32 VQVARFPYT.
  • SEQ ID No. 33 TQGSHPPYT.
  • SEQ ID No. 34 TQATRAPYS.
  • SEQ ID No. 35 FQVALFPYT.
  • SEQ ID No. 36 TQASRPMYT.
  • SEQ ID No. 37 VQVSLFPYT.
  • SEQ ID No. 38 VQLSRFPYT.
  • SEQ ID No. 40 FQSSHFPYT.
  • the heavy chain framework region of the affinity matured glypican 3 antibody comprises the amino acid sequence shown in SEQ ID No. 2-5, the affinity matured glypican 3
  • the light chain framework region of the antibody includes the amino acid sequences shown in SEQ ID No. 10-13.
  • Some embodiments of the present disclosure also provide a bispecific antibody, the bispecific antibody comprising the above-mentioned affinity matured Glypican 3 antibody.
  • the bispecific antibody has the following properties:
  • the present disclosure also provides a biological material that is any of the following B1) to B6):
  • B1) a nucleic acid molecule encoding the heavy chain and/or light chain of the antibody described in (b1) or (b2), or encoding the heavy chain and/or light chain in the antigen-binding portion of the antibody described in (b1) or (b2)
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);
  • B5 the cell line containing the nucleic acid molecule described in B1), or the cell line containing the expression cassette described in B2), or the cell line containing the recombinant vector described in B3);
  • Some embodiments of the present disclosure also provide a pharmaceutical composition, the pharmaceutical composition comprising the above-mentioned Glypican 3 antibody, the above-mentioned affinity matured Glypican 3 Antibody or any one or a combination of at least two of the above-mentioned bispecific antibodies.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or antibody fragment, bispecific antibody or antibody conjugate of the present disclosure, and optionally a pharmaceutically acceptable carrier, surfactant, diluent Any one or a combination of at least two.
  • the pharmaceutical composition comprises, in addition to an antibody, bispecific antibody or antibody conjugate of the disclosure, one or more additional therapeutic agents.
  • additional therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other Agents for the treatment of cancer.
  • Some embodiments of the present disclosure provide the above described Glypican 3 antibody, the above described affinity matured Glypican 3 antibody, the above described bispecific antibody or the above Application of the pharmaceutical composition described herein in the preparation of drugs for treating tumors.
  • the tumor comprises liver cancer.
  • Some embodiments of the present disclosure also provide the above-mentioned Glypican 3 antibody, the above-mentioned affinity matured Glypican 3 antibody, or the above-mentioned pharmaceutical composition for treating tumors the use of.
  • the tumor comprises liver cancer.
  • Some embodiments of the present disclosure also provide a method for treating tumors, the method comprising: administering the above-mentioned Glypican 3 antibody, the above-mentioned The affinity matured Glypican 3 antibody or the pharmaceutical composition described above.
  • the tumor comprises liver cancer.
  • the Glypican 3 antibody of the present disclosure can specifically bind to Glypican 3 with high affinity, and has important application value in the field of treating Glypican-3 related diseases, especially It is of great significance to the field of liver cancer drug development;
  • the present disclosure constructs an affinity maturation library by mutating the Glypican 3 antibody, which further improves the affinity of the Glypican 3 antibody;
  • the Glypican 3 antibody of the present disclosure can efficiently bind to cells overexpressing Glypican-3, activate T lymphocytes, inhibit tumor growth and promote its regression.
  • This example prepares a Glypican 3 antibody (GPC3 antibody).
  • the human GPC3 antigen is used in this disclosure, and its sequence is derived from UniProtKB:P51654.
  • the sequence of the GPC3 near-membrane antigen is derived from the 524-563 amino acids at the C-terminal of the full-length antigen.
  • the antigen sequence is cloned into a true
  • the nuclear expression vector pcDNA3.1-TEV-Fc-His was used to establish hGPC3-TEV-Fc-His recombinant eukaryotic expression plasmid, which was transfected into 293F host cells by PEI transfection method, and the cell supernatant was collected after 5 days of culture.
  • the supernatant was affinity-purified with protein A (protein A) to obtain the GPC3-TEV-Fc-His fusion protein, which was digested with TEV protease and purified by a nickel column to obtain recombinant human GPC3 extracellular region antigen and proximal membrane antigen.
  • protein A protein A
  • the human VH and human VL genes were assembled into the human scFv gene by overlapping extension PCR, and the scFv gene was cloned into the phagemid pITa vector by enzyme digestion and ligation, and transformed into host cells (TG1 Electrocompetent Cells: Lucigen.) to obtain Anti-human scFv phage library.
  • the human scFv phage library was cultured, packaged, released and purified. After three rounds of panning against recombinant human GPC3 extracellular region antigens, positive clones were enriched and sequenced. Finally, a human GPC3 antibody strain was obtained, named 10C7.
  • the human GPC3 antibody 10C7 heavy chain variable region (VH) and light chain variable region (VL) sequences presented in this disclosure are shown in Table 1, and its heavy chain framework regions VH-FR1, VH-FR2, VH-FR3, VH-FR4 has the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 respectively; Its light chain framework region VL-FR1, VL-FR2, VL- FR3 and VL-FR4 have the amino acid sequences shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13 respectively; their heavy chain complementarity determining regions HC-CDR1 and HC-CDR2 , HC-CDR3 have the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7, and SEQ ID No.8 respectively; the light chain complementarity determining regions LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid
  • the present disclosure expresses, purifies and verifies the human GPC3 antibody 10C7 described in Example 1.
  • the heavy chain and light chain DNA encoding the 10C7 antibody were synthesized, cloned into the expression vector pcDNA3.1, infected and amplified, and the plasmid was extracted, and the heavy chain and light chain of the anti-human GPC3 antibody 10C7 were transfected with PEI.
  • the chain expression plasmid was transfected into 293F cells, and after 6 days of culture, the cell culture supernatant was collected by centrifugation, and then the antibody in the supernatant was purified by Protein A affinity chromatography injection (GE healthcare).
  • the positive control antibody GC33 monoclonal antibody mimic in the present disclosure is derived from the sequence of the human-mouse chimeric antibody GC33 (patent: US 2015/0259417 A1), expressed and purified by the same method as above.
  • the human GPC3 extracellular region antigen and near-membrane antigen were diluted with NaHCO 3 buffer (pH 9.0) and then coated and blocked.
  • Antibody 10C7 and control samples (GC33 monoclonal antibody mimic and hIgG) were serially diluted, Add it to the antigen-coated well for incubation, add HRP-labeled goat anti-human IgG (H+L) secondary antibody after washing, TMB color development, the detection wavelength of the microplate reader is 450nm, read the absorbance OD value, and compare the sample concentration The value is on the X-axis, the absorbance OD value is on the Y-axis, the dose-response curve is drawn, and the EC 50 is calculated.
  • the EC 50 represents the ability of the antibody to bind to the GPC3 antigen.
  • the negative control hIgG group and the positive antibody GC33 monoclonal antibody mimic control group are set at the same time in the experiment .
  • Figure 1A is the result of ELISA detection of the binding activity of antibody 10C7 to human GPC3 extracellular domain antigen
  • Figure 1B is the result of ELISA detection of the binding activity of antibody 10C7 to human GPC3 proximal membrane antigen
  • the binding activity of antibody 10C7 to human GPC3 antigen is expressed as EC 50
  • 10C7 binds to human GPC3 extracellular domain antigen
  • EC 50 is 0.08357 ⁇ g/mL
  • 10C7 binds to human GPC3 proximal membrane antigen
  • EC 50 is 0.02791 ⁇ g/mL
  • Flow cytometry was used to detect the binding activity of antibody 10C7 to cells overexpressing human GPC3 antigen, as follows:
  • the plasmids containing the full-length human GPC3 antigen and the membrane-proximal antigen of human GPC3 were transiently transferred into 293F cells, and the 293F cells overexpressing the full-length human GPC3 antigen and the membrane-proximal antigen of human GPC3 were obtained, named For 293F/GPC3 full-length cells and 293F/GPC3 membrane-adjacent cells, collect 293F cells, 293F/GPC3 full-length cells and 293F/GPC3 membrane-adjacent cells, and pre-cool 2% FBS/PBS (diluted solution) at 4°C Wash, adjust cell density and add to 96-well plate respectively, then add diluted 10C7 antibody to 96-well plate, incubate 96-well plate at 4°C for 1h, centrifuge at 4°C to remove supernatant, wash twice, then add PE-labeled anti-human IgG Fc (PE anti-human IgG
  • an affinity maturation library of the GPC3 antibody 10C7 was prepared.
  • VH-FR1, VH-FR2, VH-FR3, and VH-FR4 in the heavy chain framework regions of the 24 affinity matured variants have SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4 respectively and the amino acid sequence shown in SEQ ID No.5; the light chain framework region VL-FR1, VL-FR2, VL-FR3, VL-FR4 have respectively as SEQ ID No.10, SEQ ID No.11, SEQ ID No.
  • This example expresses and purifies the affinity maturation variant described in example 3.
  • the heavy chain and light chain DNAs encoding the variants of affinity maturation were respectively synthesized, cloned into the expression vector pcDNA3.1, infected and amplified, the plasmid was extracted, and transfected with PEI.
  • the heavy chain and light chain expression plasmids of the affinity maturation variants were transfected into 293F cells, and after 6 days of culture, the cell culture supernatant was collected by centrifugation, and then purified by protein A (Protein A) affinity chromatography column (GE healthcare) Antibodies in the supernatant, a total of 24 affinity matured variants were obtained.
  • the ELISA method was used to detect the binding activity of the affinity matured variants to the antigen at the proximal end of the human GPC3 membrane.
  • the logarithmic value of the sample concentration was used as the X-axis, and the absorbance OD value (absorbance at 450 nm) was used as the Y-axis to plot the dose.
  • the reaction curve was used to calculate the EC 50 , which represented the ability of the sample to bind to the human GPC3 antigen.
  • the experiment set up a positive control group GC33 monoclonal antibody mimic group and a negative control group hIgG.
  • the FACS method was used to detect the binding of the 24 affinity matured variants to the cells overexpressing the full-length human GPC3 antigen and the proximal membrane end antigen, and 10C7, GC33 monoclonal antibody mimics and hIgG were used as controls, and the results As shown in Figure 4A- Figure 4G, the 24 affinity matured variants can specifically bind to 293F/GPC3 proximal end cells (solid lines on the right side of Figure 4A- Figure 4G), and the results are shown in Figure 5A- Figure 5G , the 24 affinity matured variants can specifically bind to 293F/GPC3 full-length cells (Fig. 5A-Fig. 5G right solid line).
  • the 10C7 antibody-based mutants after affinity maturation can specifically bind to the full-length human GPC3 antigen and human GPC3 proximal membrane antigen with high affinity.
  • bispecific antibodies targeting CD3 and GPC3 were constructed.
  • the recombinant eukaryotic expression plasmid of the GPC3 ⁇ CD3 bispecific antibody was constructed by means of genetic engineering technology, using the 293F cell transient expression system and the protein A affinity chromatography column Purification and other processes obtained 7 strains of GPC3 ⁇ CD3 bispecific antibodies, namely 7A7 ⁇ CD3, 5D1 ⁇ CD3, 5H9 ⁇ CD3, 8F9 ⁇ CD3, 7C6 ⁇ CD3, 7F11 ⁇ CD3 and 7F1 ⁇ CD3, antibody 10C7, positive control antibody GC33 mAb mimic.
  • the detection system composed of Jurkat/NFAT-Luc reporter gene cell line and GPC3-positive human liver cancer HepG2 cells was used to evaluate the activation effect of GPC3 ⁇ CD3 on T cells.
  • Jurkat/NFAT-Luc cell line luciferase gene Regulated by NFAT (Nuclear factor of activated T-cells) transcription factors, as effector cells
  • GPC3-positive HepG2 cells as target cells, when Jurkat/NFAT-Luc cells are co-cultured with HepG2 cells, luciferase does not express, and when When GPC3 ⁇ CD3 is added, GPC3 on HepG2 cells can activate the CD3 signaling pathway in Jurkat/NFAT-Luc cells through GPC3 ⁇ CD3, and promote the expression of luciferase, so that Jurkat can be judged by detecting the expression of luciferase in the cells.
  • the degree of activation of T cells was used to evaluate the activation effect of GPC3 ⁇ CD3 on T cells.
  • the CD3 binding arm of the bispecific antibody GPC3 ⁇ CD3 specifically binds to the CD3 complex on the surface of T cells, and the other end of the GPC3 binding arm specifically binds to the GPC3 molecule on the surface of tumor cells, enabling the formation of an immune system between T cells and tumor cells. Bridge, and then activate T cells to release cell-killing proteins such as perforin and granzyme B, thereby killing tumor cells.
  • cell-killing proteins such as perforin and granzyme B
  • lactate dehydrogenase catalyzes the dehydrogenation reaction to produce a red product, Formazan, which can produce an absorption peak at a wavelength of 490nm.
  • This disclosure uses human liver cancer cells HepG2 and Huh-7 with high GPC3 expression as target cells, and human peripheral blood mononuclear cells PBMC as effector cells to detect the ability of the seven GPC3 ⁇ CD3 bispecific antibodies to mediate the killing of tumor cells by PBMCs. lethal activity.
  • PBMC peripheral blood mononuclear cells
  • HepG2 and Huh-7 cells in good condition, and adjust the cell density respectively.
  • Beyond lactate dehydrogenase cytotoxicity kit
  • the logarithmic value of the antibody concentration is used as the abscissa, Killing activity is the ordinate, four-parameter fitting is carried out, dose-effect curve is drawn, and EC 50 value of each curve is obtained, and EC 50 represents the killing activity of GPC3 ⁇ CD3 on target cells.
  • mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • collect CT26-hGPC3 cells in the logarithmic growth phase and inoculate them at 2 ⁇ 10 6 cells/100 ⁇ L/mouse
  • the day of grouping was defined as D0 day, and the administration began on D0 day.
  • mice were negative control saline group, 7F1 ⁇ CD3
  • the 5mg/kg group and the 7F11 ⁇ CD3 5mg/kg group were given tail vein injection (iv) on D0, D3, D6 and D9 days.
  • the tumor size was observed twice a week and the mice were weighed.
  • tumor volume inhibition rate (TGI TV ): TGI TV (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100%
  • T i mean value of tumor volume of the treatment group on day i of administration
  • T 0 mean value of tumor volume of treatment group on day 0 of administration
  • V i mean value of tumor volume of the treatment group on day i of administration
  • the average tumor volume of the vehicle V 0 : the average tumor volume of the vehicle control group on the 0th day of administration).
  • the GPC3 monoclonal antibody and its affinity matured variants provided by the present disclosure can specifically bind cells expressing GPC3 antigen with high affinity, and the GPC3 ⁇ CD3 bispecific antibody can activate T lymphocytes, effectively inhibit tumor growth and Regression of tumors has important application prospects in improving immune cell activity and enhancing immune response, preventing and treating tumors and infectious diseases.
  • the present disclosure illustrates the detailed methods of the present disclosure through the above-mentioned embodiments, but the present disclosure is not limited to the above-mentioned detailed methods, that is, it does not mean that the present disclosure must rely on the above-mentioned detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement to the present disclosure, the equivalent replacement of each raw material of the disclosed product, the addition of auxiliary components, the selection of the method, etc., all fall within the scope of protection and disclosure of the present disclosure.
  • the present disclosure provides a Glypican 3 antibody and its application.
  • the present disclosure provides that the Glypican 3 antibody can specifically bind to Glypican-3 with high affinity, and can Activating T lymphocytes has important application prospects in improving immune cell activity and enhancing immune response, preventing and treating tumors and infectious diseases, and has significant applicability.

Abstract

本申请公开一种磷脂酰肌醇蛋白聚糖3抗体及其应用。所述磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列，所述磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。所述磷脂酰肌醇蛋白聚糖3抗体能够以高亲和力与磷脂酰肌醇蛋白聚糖-3特异性结合，能够激活T淋巴细胞，在提高免疫细胞活性和增强免疫应答、预防治疗肿瘤和感染性疾病等方面具有应用前景。

Description

一种磷脂酰肌醇蛋白聚糖3抗体及其应用
相关申请的交叉引用
本公开要求于2021年07月20日提交中国专利局的申请号为CN202110817631.2、名称为“一种磷脂酰肌醇蛋白聚糖3抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于肿瘤治疗和分子免疫学技术领域，涉及一种磷脂酰肌醇蛋白聚糖3抗体及其应用。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC)占原发性肝癌的70％～90％，目前治疗肝癌晚期的靶向治疗药物仅有索拉菲尼，因而研究与开发肝癌特异性靶点药物具有重要的意义。
磷脂酰肌醇蛋白聚糖3(Glypican-3，GPC3)是近年发现的肝细胞癌(hepatocellular carcinoma，HCC)细胞表面特异性膜蛋白，是HCC靶向治疗的良好靶点。GPC3是一种结合在细胞膜表面的硫酸乙酰肝素(HS)糖蛋白，其基本结构由核心蛋白、硫酸乙酰肝素链和糖基化磷脂酰肌醇3部分组成，大小约66kD，GPC3的羧基末端与糖基化磷脂酰肌醇(GPI)共价结合连接在细胞膜上，氨基末端则游离在细胞外，其内部有多个二硫键连接，使其具有球形立体结构，GPC3蛋白结构可在第358位精氨酸和第359位精氨酸处被弗林(Furin)蛋白酶切割分成两个亚基，形成1个30kD的C端亚单位和1个40kD的游离N端亚单位，其中C端亚单位可与2条硫酸乙酰肝素链(HS链)结合，残留于肿瘤细胞的膜和胞质区；N端亚单位进入循环系统后成为可溶性GPC3分子分泌入血。
GPC3的HS基可与生长因子及其受体、细胞外基质蛋白和黏附分子等相互作用，参与调节细胞增殖、分化、黏附和迁移等，GPC3主要通过Wnts信号途径发挥作用，通过自分泌和旁分泌的方式来激活经典Wnt信号途径使Wnt基因高表达，而Wnt基因的表达产物能够促进多种肿瘤组织生长。
GPC3表达特点是在肝癌组织中过表达，而在正常的人体组织中不表达或低表达，基于GPC3特有的结构和功能特点，将其作为肝癌的治疗靶点，有可能成为治疗肝癌的一把利剑。
发明内容
本公开提供一种磷脂酰肌醇蛋白聚糖3抗体，所述磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列；
所述磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
在一些实施方式中，所述磷脂酰肌醇蛋白聚糖3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列；所述磷脂酰肌醇蛋白聚糖3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
本公开还提供一种亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体由上述磷脂酰肌醇蛋白聚糖3抗体经过突变处理制备得到。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.14所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.20-40中任意一种所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.17-19中任意一种所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.16所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链框架区包括如SEQ ID No.2-5所示的氨基酸序列；所述抗体的轻链框架区包括SEQ ID No.10-13所示的氨基酸序列。
本公开还提供生物材料，所述生物材料为以下B1)至B6)中的任一种：
B1)编码(b1)或(b2)所述抗体或者(b1)或(b2)所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)上文所述的磷脂酰肌醇蛋白聚糖3抗体，(b2)上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
B6)编码(b1)或(b2)所述抗体或者(b1)或(b2)所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。
本公开还提供一种药物组合物，所述药物组合物包括所述磷脂酰肌醇蛋白聚糖3抗体和/或上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体。
在一些实施方式中，所述药物组合物还包含一种或多种治疗剂。
在一些实施方式中，所述治疗剂包括以下的至少一种：化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂。
本公开还提供所述磷脂酰肌醇蛋白聚糖3抗体、上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或所述的药物组合物在制备治疗肿瘤药物中的应用。
在一些实施方式中，所述肿瘤包括肝癌。
本公开还提供所述磷脂酰肌醇蛋白聚糖3抗体、上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或所述药物组合物，用于治疗肿瘤的用途。
在一些实施方式中，所述肿瘤包括肝癌。
本公开还提供一种治疗肿瘤的方法，所述方法包括：向所述有此需要的受试者给药所述磷脂酰肌醇蛋白聚糖3抗体、上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或所述药物组合物。
在一些实施方式中，所述肿瘤包括肝癌。
附图说明
图1A为10C7抗体与GPC3胞外区抗原的结合活性图；
图1B为10C7抗体与GPC3近膜端抗原的结合活性图；
图2A为10C7抗体与293F/GPC3全长细胞结合活性图；
图2B为10C7抗体与293F/GPC3近膜端细胞结合活性图；
图3A为亲和力成熟变体(5D1、8G2、5H9和8H5)与GPC3近膜端抗原的结合活性图；
图3B为亲和力成熟变体(5C1、7F11和5H1)与GPC3近膜端抗原的结合活性图；
图3C为亲和力成熟变体(7H3、11C6、7A2和1A6)与GPC3近膜端抗原的结合活性图；
图3D为亲和力成熟变体(5A5、8F1、5E11和5B6)与GPC3近膜端抗原的结合活性图；
图3E为亲和力成熟变体(5A4、5D8、5B10和5B8)与GPC3近膜端抗原的结合活性图；
图3F为亲和力成熟变体(7C6、8F9、9G4和7F1)与GPC3近膜端抗原的结合活性图；
图3G为亲和力成熟变体7A7与GPC3近膜端抗原的结合活性图；
图4A为FACS检测抗体(hIgG、GC33单抗模拟物、5D1和8G2)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4B为FACS检测抗体(5H9、8H5、5C1和7F11)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4C为FACS检测抗体(5H1、7H3、11C6和7A2)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4D为FACS检测抗体(1A6、5A5、8F1和5E11)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4E为FACS检测抗体(5B6、5A4、5D8和5B10)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4F为FACS检测抗体(5B8、7C6、8F9和9G4)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图4G为FACS检测抗体(7F1、7A7)与过表达人GPC3近膜端抗原的细胞的结合活性图；
图5A为FACS检测抗体(hIgG、GC33单抗模拟物、5D1和8G2)与过表达人GPC3全长抗原的细胞的结合活性图；
图5B为FACS检测抗体(5H9、8H5、5C1和7F11)与过表达人GPC3全长抗原的细胞的结合活性图；
图5C为FACS检测抗体(5H1、7H3、11C6和7A2)与过表达人GPC3全长抗原的细胞的结合活性图；
图5D为FACS检测抗体(1A6、5A5、8F1和5E11)与过表达人GPC3全长抗原的细胞的结合活性图；
图5E为FACS检测抗体(5B6、5A4、5D8和5B10)与过表达人GPC3全长抗原的细胞的结合活性图；
图5F为FACS检测抗体(5B8、7C6、8F9和9G4)与过表达人GPC3全长抗原的细胞的结合活性图；
图5G为FACS检测抗体(7F1、7A7)与过表达人GPC3全长抗原的细胞的结合活性图；
图6A为GPC3×CD3双特异性抗体(5H9×CD3、10C7×CD3、5D1×CD3和hIgG)对T细胞激活作用检测结果图；
图6B为GPC3×CD3双特异性抗体(7C6×CD3、7A7×CD3、8F9×CD3、7F11×CD3和7F1×CD3)对T细胞激活作用检测结果图；
图7A为GPC3×CD3双特异性抗体(7C6×CD3、7F11×CD3、7F1×CD3、7A7×CD3、8F9×CD3、5D1×CD3和5H9×CD3)对HepG2细胞的杀伤作用检测结果图；
图7B为GPC3×CD3双特异性抗体(7C6×CD3、7F11×CD3、7F1×CD3、7A7×CD3、8F9×CD3、5D1×CD3和5H9×CD3)对Huh-7细胞的杀伤作用检测结果图；
图8为BALB/c-hCD3E小鼠皮下CT26-hGPC3肿瘤体积变化图。
具体实施方式
为进一步阐述本公开所采取的技术手段及其效果，以下结合实施例和附图对本公开作进一步地说明。可以理解的是，此处所描述的实施方式仅仅用于解释本公开，而非对本公开的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
除非本文另有定义，与本公开结合使用的科学和技术术语及其缩略语应具有本公开所属领域的普通技术人员通常理解的含义。以下列举了本文中使用的部分术语和缩略语。
抗体：antibody，Ab。
免疫球蛋白：immunoglobulin，Ig。
重链：heavy chain，HC。
轻链：light chain，LC。
重链可变区：heavy chain variable domain，VH。
重链恒定区：heavy chain constant domain，CH。
轻链可变区：light chain variable domain，VL。
轻链恒定区：light chain constant domain，CL。
抗原结合区：antigen binding fragment，Fab。
铰链区：hinge region。
Fc片段：fragment crystallizable region，Fc region。
单克隆抗体：monoclonal antibodies，mAbs。
抗体依赖性细胞毒作用：antibody-dependent cell-mediated cytotoxicity，ADCC。
补体依赖性细胞毒性作用：complement dependent cytotoxicity，CDC。
自然杀伤细胞：natural killing cell，NK细胞。
双特异性抗体：bispecific antibody，BsAb。
T细胞受体：T cell receptor，TCR。
主要组织相容性复合体：major histocompatibility complex，MHC。
互补决定区：complementarity determining region，CDR，是指抗体的抗原互补结合区。
免疫受体酪氨酸活化基序：immunoreceptor tyrosine-based activation motif，ITAM。
单链可变区抗体片段(又称单链抗体)：single-chain variable fragment，scFv；
过继免疫治疗：adoptive cellular immunotherapy，ACI。
淋巴因子激活的杀伤细胞：lymphokine-activated killer cell，LAK细胞。
肿瘤浸润淋巴细胞：Tumor Infiltrating Lymphocyte，TIL细胞。
细胞因子诱导的杀伤细胞：cytokine-induced killer cell，CIK细胞。
本公开所述分子克隆、细胞培养、蛋白纯化、免疫学实验、微生物学、动物模型等试验的操作步骤为该领域内被广泛应用的常规步骤。除非上下文另有说明，否则本公开单数术语包含复数且复数含义包括单数含义。除非另外指明，否则本公开所述核苷酸序列按照5’端至3’端的方向从左至右排列并书写。除非另外指明，否则本公开所述氨基酸序列按照从氨基端(N末端)到羧基端(C末端)的方向从左至右排列并书写。本公开所提及的氨基酸三字母缩写及核苷酸单字母缩写为该技术领域普遍接受的形式，氨基酸单字母缩写为IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的形式。
术语“氨基酸”是指20种天然存在的氨基酸之一或可能存在于特定的限定位置的任何非天然类似物。本公开所述“氨基酸突变”是指多肽序列中的氨基酸取代、插入、缺失和修饰，以及氨基酸取代、插入、缺失和修饰的任意组合。本文中可选的氨基酸修饰是取代。本公开中“氨基酸取代”或“取代”是指将亲本多肽序列中特定位置的氨基酸替代为另一种氨基酸。例如，取代C220S是指变体多肽，其中多肽的位置220处的氨基半胱氨酸已被氨基酸丝氨酸替代。氨基酸突变可以通过分子克隆或化学的方法实现，分子克隆的方法包括PCR、定点突变、全基因合成等。
术语“蛋白”、“肽链”、“多肽链”是指两个以上的氨基酸通过肽键连接的分子，包含天然蛋白、人工蛋白、蛋白片段、突变蛋白和融合蛋白等。
术语“结构域”是指生物大分子中具有独立功能的特异结构区域，结构域具有独立的三级结构，其功能不依赖于生物大分子中的其余部分，本公开中的结构域特指蛋白中这样的区域，如重链可变区VH结构域、轻链可变区VL结构域，结构域之间相互结合可以构成一个大的结构域。
术语“抗体”指包含至少一个抗原识别位点并能特异性结合抗原的免疫球蛋白分子。在此，术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质，如蛋白质、多肽、肽、碳水化合物、多聚核苷酸、脂质、半抗原或上述物质的组合。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。本公开所提及的术语“抗体”包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同表位结合结构域的多特异性抗体(例如，双特异性抗体)、人抗体、人源化抗体、翻译后修饰抗体、骆驼抗体、嵌合抗体、包含抗体抗原决定簇的融合蛋白、以及包含抗原识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合位点的分子。
术语“Fab”、“Fab区域”、“Fab片段”或“Fab分子”是抗原结合片段，包含免疫球蛋白重链的VH结构域、CH1结构域以及轻链的VL结构域、CL结构域，重链第一个恒定区结构域CH1与轻链的恒定区结构域CL结合，重链的可变区结构域VH和轻链的可变区结构域VL结合。
术语“Fc”、“Fc区域”、“Fc片段”或“Fc分子”是抗体的效应区，能够引起如CDC、ADCC、ADCP、细胞因子释放等。天然的抗体Fc通常是由两个相同的蛋白片段结合构成，该蛋白片段包含两个或三个免疫球蛋白恒定区结构域。本公开中所述Fc包含天然Fc以及突变的Fc。虽然Fc区的边界可以变化，但人IgG重链Fc区通常定义为包含从C226或P230开始到其羧基末端的残基。在实验条件下，免疫球蛋白单体经木瓜蛋白酶酶切生成的片段分别为Fab和Fc。抗体的“铰链”或“铰链区”是指包含抗体的第一和第二恒定结构域(CH1和CH2)之间的氨基酸的柔性多肽。
除非另有说明，否则本公开所述抗体可变区氨基酸编号使用Kabat等人在1991年所阐述的编码方案，即“Kabat索引”或“Kabat编号”(Kabat,E.A.et al.Sequences of Proteins of Immunological Interest,5th ed.,NIH Publication No.91–3242,Bethesda,MD.:1991)。除非另有说明，否则本公开所述抗体恒定区氨基酸编号使用EU索引(Edelman GM,et.al.Proc Natl Acad Sci U S A 1969,63:78-85.)。
术语“抗原结合位点”指抗原结合分子与抗原产生直接相互作用的一个或多个氨基酸残基，抗体的抗原结合位点由抗原互补决定区(CDR)构成，天然免疫球蛋白分子通常包含两个抗原结合位点，Fab分子通常包含一个抗原结合位点。
术语“T细胞激活”指T淋巴细胞尤其是杀伤性T淋巴细胞的一种或多种免疫反应，包括：增值、分化、细胞因子的释放、杀伤性效应分子的分泌、细胞杀伤等。
术语“EC 50”即半最大效应浓度(concentration for 50％of maximal effect)，是指引起50％最大效应所对应的抗体浓度。
本文所用“特异性结合”是指，两种分子间的非随机的结合反应，如抗体及其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M、或10-10M或更小的亲和力(KD)结合该抗原。在本公开的一些实施方式中，术语“靶向”指特异性结合。
本文所用“KD”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、 10-7M、10-8M、10-9M、或10-10M或更小的平衡解离常(KD)结合抗原。
术语“单链可变区抗体片段”或“scFv”指免疫球蛋白重链可变区VH和轻链可变区VL的融合蛋白，包括N端为VH以及N端为VL的不同组合形式，利用构建重组蛋白的常规分子克隆方法即可制备(Sambrook JF,E.F.et al.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York:2012)。
术语“人源化抗体”是指人源免疫球蛋白(受体抗体)的部分或者全部CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或者抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或者反应性的非人源(如小鼠、大鼠或兔)抗体。此外，受体抗体的架构区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或者被其他抗体的氨基酸残基替换，以进一步改善或者优化抗体的一种或多种特性。
术语“宿主细胞”指导入外源核酸的细胞及其后代，可通过编码多肽的核苷酸转化或转染，从而表达外源多肽。本公开所述的宿主细胞包括但不限于CHO细胞(Chinese hamster ovary cells，中国仓鼠卵巢细胞)、HEK293细胞(Human embryonic kidney cells 293，人胚肾细胞293)、BHK细胞(Baby Hamster Kidney，幼仓鼠肾细胞)、骨髓瘤细胞、酵母、昆虫细胞或原核细胞如大肠杆菌(Escherichia coli)等。应当指出，本公开所述“宿主细胞”不仅指导入了外源核酸的细胞，同时还包括了该细胞的后代，由于在细胞分裂过程中后代细胞会发生突变，但仍属于本公开所述的术语范围。
本公开还进一步包含编码这些多肽链的核酸序列。在表达抗体的过程中，将核酸序列插入合适的载体中，载体包括但不限于：质粒、噬菌表达载体、柯斯质粒、人工染色体、噬菌体以及动物病毒。表达载体中包含用于调控表达的元件，包括但不限于启动子、转录起始序列、增强子、信号肽序等。启动子包括但不限于T7启动子、T3启动子、SP6启动子、β-actin启动子、EF-1α启动子、CMV启动子以及SV40启动子。将表达载体转入宿主细胞中可使用本领域内已知的合适方法，包括但不限于：磷酸钙沉淀法、聚乙烯亚胺转染法、脂质体转染法、电穿孔法、PEI(聚乙烯亚胺)转染法。
如本文所用，“有效量”可以指本文所述的组合物或本文所述的药物制剂的量，其将引起研究者、兽医、医师或其他临床医生正在寻求的组织、系统、动物、植物、原生动物、细菌、酵母或人类的期望的生物学或医学响应。
如本文所用，术语“受试者”是指脊椎动物，可选地为哺乳动物，可选地为人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
应当理解本文所用术语仅用于描述本公开上下文诸如实施方式和实施例的目的，并不旨在限制本发明的范围。
本公开一些实施方式提供一种磷脂酰肌醇蛋白聚糖3抗体及其应用，所述磷脂酰肌醇蛋白聚糖3抗体能够以高亲和力与磷脂酰肌醇蛋白聚糖-3特异性结合，能够激活T淋巴细胞，在提高免疫细胞活性和增强免疫应答、预防治疗肿瘤和感染性疾病等方面具有重要的应用前景。
本公开的一些实施方式提供一种磷脂酰肌醇蛋白聚糖3抗体，所述磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区(VH-CDR1、VH-CDR2和VH-CDR3)包括SEQ ID No.6-8所示的氨基酸序列，所述磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区(VL-CDR1、VL-CDR2和VL-CDR3)包括SEQ ID No.14-16所示的氨基酸序列。
根据本公开，所述磷脂酰肌醇蛋白聚糖3抗体能够以高亲和力与磷脂酰肌醇蛋白聚糖-3特异性结合，能够激活T淋巴细胞，在提高免疫细胞活性和增强免疫应答、预防治疗肿瘤和感染性疾病等方面具有重要的应用前景。
根据本公开，所述磷脂酰肌醇蛋白聚糖3抗体具有以下性质：
a.特异性结合人GPC3全长抗原、人GPC3近膜端抗原；
b.特异性结合过表达人GPC3全长抗原、人GPC3近膜端抗原的细胞。
在一些实施方式中，所述磷脂酰肌醇蛋白聚糖3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列，所述磷脂酰肌醇蛋白聚糖3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
在一些实施方式中，所述磷脂酰肌醇蛋白聚糖3抗体的重链框架区包括如SEQ ID No.2-5所示的氨基酸序列，所述磷脂酰肌醇蛋白聚糖3抗体的轻链框架区包括SEQ ID No.10-13所示的氨基酸序列。
SEQ ID No.1：
Figure PCTCN2022106547-appb-000001
SEQ ID No.2：QVQLVQSGAEVKKPGASVKLSCKAS。
SEQ ID No.3：MHWVKQTPGKGLKWIGA。
SEQ ID No.4：AYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYC。
SEQ ID No.5：WGQGTLVTVSS。
SEQ ID No.6(VH-CDR1)：GYTFTDYE。
SEQ ID No.7(VH-CDR2)：LDPKTGDT。
SEQ ID No.8(VH-CDR3)：TRFYSYTY。
SEQ ID No.9：
Figure PCTCN2022106547-appb-000002
SEQ ID No.10：DIVLTQSPLSLTVTPGEPASISCRSS。
SEQ ID No.11：LSWLQQRPGQPPRLLIY。
SEQ ID No.12：NRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC。
SEQ ID No.13：FGQGTKVEIK。
SEQ ID No.14(VL-CDR1)：ESLVHSNGNTY。
SEQ ID No.15(VL-CDR2)：KIS。
SEQ ID No.16(VL-CDR3)：TQATQFPYT。
本公开的一些实施方式还提供编码上述磷脂酰肌醇蛋白聚糖3抗体的DNA片段。
在一些实施方式中，所述磷脂酰肌醇蛋白聚糖3抗体的轻链具有SEQ ID NO.41所示的核苷酸序列，所述磷脂酰肌醇蛋白聚糖3抗体的重链具有SEQ ID NO.42的核苷酸序列。
SEQ ID NO.41：
Figure PCTCN2022106547-appb-000003
SEQ ID NO.42：
Figure PCTCN2022106547-appb-000004
本公开的一些实施方式还提供一种表达载体，表达载体包含至少一个拷贝的如上所述的磷脂酰肌醇蛋白聚糖3抗体的DNA片段。
本公开的一些实施方式还提供一种宿主细胞，宿主细胞包含如上所述的表达载体。
本公开的一些实施方式还提供一种亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，亲和力成熟磷脂酰肌醇蛋白聚糖3抗体由上文所述的磷脂酰肌醇蛋白聚糖3抗体经过突变处理制备得到。
在一些实施方式中，突变处理包括对磷脂酰肌醇蛋白聚糖3抗体的轻链可变区和/或重链可变区进行随机突变。例如，在一些实施方式中，磷脂酰肌醇蛋白聚糖3抗体的轻链可变区和/或重链可变区经随机突变得到的突变如表2所述。据信，不受理论的约束，所述随机突变进一步提高了磷脂酰肌醇蛋白聚糖3抗体的亲和力。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.14所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.20-40中任意一种所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.17-19中任意一种所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.16所示的氨基酸序列。
SEQ ID No.17：HPLLHSNGNTY。
SEQ ID No.18：ESLVHINGNTY。
SEQ ID No.19：QSLLSSNGNTY。
SEQ ID No.20：TQSAFAPYT。
SEQ ID No.21：VQVSFFPYT。
SEQ ID No.22：TQLTRVPYT。
SEQ ID No.23：FQNGRFPYT。
SEQ ID No.24：TQATRPPYS。
SEQ ID No.25：VQVSSFPYT。
SEQ ID No.26：TQATHLYYS。
SEQ ID No.27：TQATRPPYL。
SEQ ID No.28：VQVSAFPYT。
SEQ ID No.29：LQVSIFPYT。
SEQ ID No.30：VQVSRFPYT。
SEQ ID No.31：VQTATFPYT。
SEQ ID No.32：VQVARFPYT。
SEQ ID No.33：TQGSHPPYT。
SEQ ID No.34：TQATRAPYS。
SEQ ID No.35：FQVALFPYT。
SEQ ID No.36：TQASRPMYT。
SEQ ID No.37：VQVSLFPYT。
SEQ ID No.38：VQLSRFPYT。
SEQ ID No.39：VQVSMFPYT。
SEQ ID No.40：FQSSHFPYT。
在一些实施方式中，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链框架区包括如SEQ ID No.2-5所示的氨基酸序列，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链框架区包括SEQ ID No.10-13所示的氨基酸序列。
本公开的一些实施方式还提供一种双特异性抗体，所述双特异性抗体包括上文所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体。
根据本公开，所述双特异性抗体具有以下性质：
a.在GPC3阳性的靶细胞存在时，对T细胞有激活作用；
b.介导T细胞杀伤GPC3阳性的肿瘤细胞；
c.体内抑制GPC3阳性肿瘤的生长。
本公开还提供生物材料，所述生物材料为以下B1)至B6)中的任一种：
B1)编码(b1)或(b2)所述抗体的重链和/或轻链的核酸分子，或者编码(b1)或(b2)所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)上文所述的磷脂酰肌醇蛋白聚糖3抗体，(b2)上文任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
B6)编码(b1)或(b2)所述抗体的重链可变区和/或轻链可变区的核酸分子，或者编码(b1)或(b2)所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。本公开的一些实施方式还提供一种药物组合物，所述药物组合物包括上文所述的磷脂酰肌醇蛋白聚糖3抗体、上文所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或上文所述的双特异性抗体中的任意一种或至少两种的组合。
在一些实施方式中，本公开涉及药物组合物，其包含本公开的抗体或抗体片段、双特异性抗体或抗体偶联物，以及任选地药学上可接受的载体、表面活性剂、稀释剂的任意一种或至少两种的组合。在一些实施方式中，除本公开的抗体、双特异性抗体或抗体偶联物外，所述药物组合物还包含一种或多种另外的治疗剂。在一些实施方式中，所述另外的治疗剂包括但不限于化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂和其它治疗癌症的试剂。
本公开的一些实施方式提供上文所述的磷脂酰肌醇蛋白聚糖3抗体、上文所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体、上文所述的双特异性抗体或上文所述的药物组合物在制备治疗肿瘤药物中的应用。
在一些实施方式中，所述肿瘤包括肝癌。
本公开的一些实施方式还提供上文所述磷脂酰肌醇蛋白聚糖3抗体、上文所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或上文所述药物组合物，用于治疗肿瘤的用途。
在一些实施方式中，所述肿瘤包括肝癌。
本公开的一些实施方式还提供一种治疗肿瘤的方法，所述方法包括：向所述有此需要的受试者给药上文所述磷脂酰肌醇蛋白聚糖3抗体、上文所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或上文所述药物组合物。
在一些实施方式中，所述肿瘤包括肝癌。
本公开的磷脂酰肌醇蛋白聚糖3抗体能够以高亲和力与磷脂酰肌醇蛋白聚糖3特异性结合，在治疗磷脂酰肌醇蛋白聚糖-3相关的病症领域具有重要应用价值，特别是对于肝癌药物开发领域具有重要意义；
本公开通过对所述磷脂酰肌醇蛋白聚糖3抗体进行突变构建亲和力成熟文库，进一步提高了磷脂酰肌醇蛋白聚糖3抗体的亲和力；
本公开的磷脂酰肌醇蛋白聚糖3抗体能够高效地与过表达磷脂酰肌醇蛋白聚糖-3的细胞结合，能够激活T淋巴细胞，抑制肿瘤生长并促进其消退。
实施例
实施例1
本实施例制备一种磷脂酰肌醇蛋白聚糖3抗体(GPC3抗体)。
(1)人GPC3抗原的制备
本公开中采用人GPC3抗原，序列来源于UniProtKB:P51654，GPC3近膜端抗原的序列来源于全长抗原C-端524～563位的氨基酸，本公开在此基础上，将抗原序列克隆至真核表达载体pcDNA3.1-TEV-Fc-His，建立hGPC3-TEV-Fc-His重组真核表达质粒，以PEI转染法转染至293F宿主细胞，经5天培养后收集细胞上清液，上清经蛋白A(protein A)亲和纯化得到GPC3-TEV-Fc-His融合蛋白，再利用TEV蛋白酶酶切，镍柱纯化后得到重组人GPC3胞外区抗原及近膜端抗原。
(2)人源抗GPC3抗体的获得
取人外周血，分离单核细胞，提取总RNA，用逆转录试剂盒提取得到cDNA，以该cDNA为模板，参考LimT.S.等(参见：Lim T S,Mollova S,Rubelt F,et al.An optimised procedure for amplification of rearranged human antibody genes of different isotypes[J].New Biotechnology,2010,27(2):108-117.)设计特异引物进行PCR，PCR扩增其VL片段，获得人源VH和人源VL基因。
将人源VH和人源VL基因，通过重叠延伸PCR，组装成人源scFv基因，利用酶切连接将scFv基因克隆至噬菌粒pITa载体，转化至宿主细胞(TG1 Electrocompetent Cells：Lucigen.)，获得抗人scFv噬菌体文库。
人scFv噬菌体文库经过培养、包装、释放及纯化，针对重组人GPC3胞外区抗原，经三轮淘洗后，富集阳性克隆，测序，最终获得1株人源GPC3抗体，命名为10C7。
本公开呈现的人源GPC3抗体10C7重链可变区(VH)和轻链可变区(VL)序列如表1所示，其重链框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4具有分别如SEQ ID No.2、SEQ ID No.3、SEQ ID No.4和SEQ ID No.5所示的氨基酸序列；其轻链框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4具有分别如SEQ ID No.10、SEQ ID No.11、SEQ ID No.12和SEQ ID No.13所示的氨基酸序列；其重链互补决定区HC-CDR1、HC-CDR2、HC-CDR3具有分别如SEQ ID No.6、SEQ ID No.7和SEQ ID No.8所示的氨基酸序列；轻链互补决定区LC-CDR1、LC-CDR2、LC-CDR3具有分别如SEQ ID No.14、SEQ ID No.15和SEQ ID No.16所示的氨基酸序列。
表1
Figure PCTCN2022106547-appb-000005
Figure PCTCN2022106547-appb-000006
实施例2
本公开对实施例1所述人源GPC3抗体10C7进行表达、纯化及验证。
(1)10C7的构建和表达
分别合成编码10C7抗体重链和轻链DNA，并将其克隆至表达载体pcDNA3.1中，感染并扩增，并提取质粒，用PEI转染法，将抗人GPC3抗体10C7的重链和轻链表达质粒转染至293F细胞中，培养6天后，离心收集细胞培养上清，随后用Protein A亲和层析注(GE healthcare)纯化上清液中的抗体。
本公开中的阳性对照抗体GC33单抗模拟物，序列来源于人鼠嵌合抗体GC33(专利：US 2015/0259417 A1)，采用上述同样方法表达、纯化。
(2)10C7与人GPC3抗原的结合活性
采用ELISA法检测抗人GPC3抗体10C7与人GPC3抗原的结合活性，方法如下：
将人GPC3胞外区抗原及近膜端抗原分别用NaHCO 3缓冲液(pH 9.0)稀释后进行包被，封闭，将抗体10C7以及对照样品(GC33单抗模拟物和hIgG)分别进行梯度稀释，加入到抗原包被孔进行孵育，洗涤后加入HRP标记的山羊抗人IgG(H+L)二抗，TMB显色，酶标仪检测波长为450nm，读取吸光度OD值，以样品浓度的对数值为X轴，吸光度OD值为Y轴，绘制剂量反应曲线，计算EC 50，以EC 50代表抗体与GPC3抗原结合的能力，实验同时设置阴性对照hIgG组和阳性抗体GC33单抗模拟物对照组。
图1A为ELISA检测抗体10C7与人GPC3胞外区抗原的结合活性结果，图1B为ELISA检测抗体10C7与人GPC3近膜端抗原的结合活性结果，抗体10C7与人GPC3抗原的结合活性，以EC 50来表示，10C7与人GPC3胞外区抗原结合，EC 50为0.08357μg/mL，10C7与人GPC3近膜端抗原结合，EC 50为0.02791μg/mL，表明本公开的GPC3抗体10C7能与人GPC3胞外区抗原以及GPC3近膜端抗原结合。
(3)抗体10C7与过表达人GPC3抗原细胞的结合活性
采用流式细胞术(FACS)检测抗体10C7与过表达人GPC3抗原细胞的结合活性，方法如下：
以PEI转染法，分别将包含人GPC3全长抗原和人GPC3近膜端抗原的质粒瞬转至293F细胞中，获得过表达人GPC3全长抗原和人GPC3近膜端抗原的293F细胞，命名为293F/GPC3全长细胞和293F/GPC3近膜端细胞，收集293F细胞、293F/GPC3全长细胞和293F/GPC3近膜端细胞，以4℃预冷的2％FBS/PBS(稀释液)洗涤，调整细胞密度分别加入96孔板中，再将稀释后的10C7抗体，加入96孔板中，将96孔板置于4℃孵育1h，4℃离心去上清，洗涤两次，再加入PE标记的抗人IgG Fc(PE anti-human IgG Fc,Invitrogen)，4℃避光孵育1h，4℃离心去上清，洗涤两次，最后用稀释液重悬细胞后立即流式细胞仪上机检测。
结果见图2A和图2B，可见抗体10C7与293F细胞不结合，但可特异性结合293F/GPC3全长细胞(图2A)及293F/GPC3近膜端细胞(图2B)，表明本公开的GPC3抗体10C7能与过表达人GPC3抗原细胞的结合。
实施例3
本实施例制备GPC3抗体10C7亲和力成熟文库。
由图1A可知，本公开制备的GPC3抗体10C7与GPC3全长抗原的结合活性稍弱于人鼠嵌合抗体GC33单抗模拟物，为进一步获得与GPC3抗原有高亲和力的抗体，本实施例对抗体10C7轻链的可变区CDR1、CDR2以及CDR3分别进行随机突变来提升亲和力。
(1)亲和力成熟文库的生成
为了生成10C7的互补决定区CDR 1、CDR 2、CDR3中随机化的亲和力成熟文库，将编码CDR 1、CDR 2、CDR3中的氨基酸三联密码子随机化，根据KontermannR等(参见：Kontermann R,S Dübel.Antibody Engineering||Synthetic Antibody Libraries[J].2010,10.1007/978-3-642-01144-3(Chapter 6):85-97.)方法分别合成CDR 1、CDR 2、CDR3的三联密码子上下游随机引物，使用10C7的单链可变区抗体形式(VH-linker-VL)作为模板，使用抗体上游引物及下游随机引物扩增第一片段(片段1)，使用相同模板，使用抗体下游引物及上游随机引物扩增第二片段(片段2)，片段1的3'端与片段2的5'端具有交叠区，交叠延伸PCR生成具有随机化CDR的第三片段(片段3)，然后，将所得的片段3克隆至噬菌粒载体经电转化大肠杆菌感受态细胞(TG1 Electrocompetent Cells：Lucigen.)，并均匀涂布平板，37℃过夜培养后收集菌体，所得菌体为人源化抗体的亲和力成熟文库。
(2)亲和力成熟文库的筛选
利用噬菌体展示技术实施亲和力成熟的人源化抗体的生成，如前述实施例方法，亲和力成熟文库噬菌体文库经包装、展示、三轮淘洗后，富集阳性克隆并测序，得到24个亲和力成熟变体，其中，24个亲和力成熟变体中重链框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4分别具有如SEQ ID No.2、SEQ ID No.3、SEQ ID No.4和SEQ ID No.5所示的氨基酸序列；轻链框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4分别具有如SEQ ID No.10、SEQ ID No.11、SEQ ID No.12和SEQ ID No.13所示的氨基酸序列；重链互补决定区HC-CDR 1、HC-CDR 2、HC-CDR 3分别具有如SEQ ID No.6、SEQ ID No.7、SEQ ID No.8所示的氨基酸序列，各个变体的轻链互补决定区LC-CDR 1、LC-CDR 2和LC-CDR 3区序列如表2所示。
表2
Figure PCTCN2022106547-appb-000007
实施例4
本实施例表达并纯化实施例3所述亲和力成熟变体。
按实施例2所述方法，分别合成编码亲和力成熟各变体的重链和轻链DNA，且将其克隆至表达载体pcDNA3.1中，感染并扩增，提取质粒，用PEI转染法，将亲和力成熟各变体的重链和轻链表达质粒转染至293F细胞中，培养6天后，离心收集细胞培养上清，随后用蛋白A(Protein A)亲和层析柱(GE healthcare)纯化上清液中的抗体，共获得24株亲和力成熟变体。
实施例5
本实施例对实施例4制备的亲和力成熟各变体进行功能验证。
(1)亲和力成熟变体与人GPC3抗原的结合活性
如前实施例所述，采用ELISA方法检测亲和力成熟变体与人GPC3近膜端抗原的结合活性，以样品浓度的对数值为X轴，吸光度OD值(450nm处吸光度)为Y轴，绘制剂量反应曲线，计算EC 50，以EC 50代表样品与人GPC3抗原结合的能力，实验设置阳性对照组GC33单抗模拟物组以及阴性对照组hIgG。
结果如图3A-图3G所示，24个亲和力成熟变体均可以与人GPC3近膜端抗原结合，其结合活性如表3所示，可知基于抗体10C7突变得到的24个亲和力成熟变体均具备与GPC3近膜端抗原的高亲和力。
表3
抗体名称 EC 50(μg/mL) 抗体名称 EC 50(μg/mL)
GC33单抗模拟物 0.06296 5A5 0.09507
10C7 0.1414 8F1 0.4317
5D1 0.03233 5E11 0.1199
8G2 0.08799 5B6 0.09115
5H9 0.02253 5A4 0.1467
8H5 0.05705 5D8 0.04277
5C1 0.07600 5B10 0.05373
7F11 0.02830 5B8 0.5119
5H1 0.08515 7C6 0.04278
7H3 0.1960 8F9 0.04495
11C6 0.08966 9G4 0.06709
7A2 0.06700 7F1 0.03629
1A6 0.1807 7A7 0.1013
(2)亲和力成熟变体与过表达人GPC3抗原细胞的结合
如前实施例所述，采用FACS法检测所述24个亲和力成熟变体与过表达人GPC3全长抗原和近膜端抗原细胞的结合，以10C7、GC33单抗模拟物及hIgG作为对照，结果如图4A-图4G所示，所述24个亲和力成熟变体均可特异性结合293F/GPC3近膜端细胞(图4A-图4G右侧实线)，结果如图5A-图5G所示，所述24个亲和力成熟变体均可特异性结合293F/GPC3全长细胞(图5A-图5G右侧实线)。
综上所述，基于10C7抗体进行突变亲和力成熟后的变体均能以高亲和力特异性结合人GPC3全长抗原和人GPC3近膜端抗原。
实施例6
本实施例构建靶向CD3和GPC3的双特异性抗体。
根据前述实施例5的结果，选取与人GPC3近膜端抗原结合活性较强的7株亲和力成熟变体(7A7、5D1、5H9、8F9、7C6、7F11和7F1)，构建靶向CD3和GPC3的双特异性抗体。
基于本发明人公开专利(CN107106682A)所述的技术，通过基因工程技术手段，构建GPC3×CD3双特异性抗体的重组真核表达质粒，利用293F细胞瞬转表达系统，蛋白A亲和层析柱纯化等过程获得7株GPC3×CD3双特异性抗体，分别为7A7×CD3、5D1×CD3、5H9×CD3、8F9×CD3、7C6×CD3、7F11×CD3和7F1×CD3，抗体10C7、阳性对照抗体GC33单抗模拟物。
采用下述方法对获得的7株GPC3×CD3双特异性抗体进行生物学活性的验证。
(1)检测GPC3×CD3双特异性抗体对T细胞的激活作用
采用Jurkat/NFAT-Luc报告基因细胞系和GPC3阳性的人肝癌细胞HepG2细胞组成的检测系统，来评价GPC3×CD3对T细胞的激活作用，在Jurkat/NFAT-Luc细胞系中，荧光素酶基因受NFAT(Nuclear factor of activated T-cells)转录因子调控，作为效应细胞，GPC3阳性的HepG2细胞作为靶细胞，当Jurkat/NFAT-Luc细胞与HepG2细胞共培养时，荧光素酶不表达，而当加入GPC3×CD3时，HepG2细胞上的GPC3可以通过GPC3×CD3激活Jurkat/NFAT-Luc细胞内的CD3信号通路，促使荧光素酶表达，从而可以通过检测细胞中的荧光素酶表达量来判断Jurkat T细胞激活程度的强弱。
实验中，在96孔细胞培养板中依次加入适量HepG2细胞、Jurkat/NFAT-Luc细胞，再将上述7株GPC3×CD3双特异性抗体分别进行梯度稀释，将不同浓度的GPC3×CD3双特异性抗体加入到上述细胞培养板中，37℃静置培养6h后，加入ONE-Glo荧光素酶(Luciferase)检测试剂，使用多功能酶标仪测定化学发光值，以抗体浓度的对数值为横坐标，平均化学发光值为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC 50值，以EC 50代表GPC3×CD3激活T细胞的活性，实验设置同型对照抗体hIgG组(hIgG抗体梯度稀释方式与上述7株GPC3×CD3双特异性抗体相同)。
结果如图6A和图6B所示，当与GPC3阳性肿瘤细胞HepG2、Jurkat/NFAT-Luc细胞共同孵育时，本公开构建的7株GPC3×CD3双特异性抗体(7A7×CD3、5D1×CD3、5H9×CD3、8F9×CD3、7C6×CD3、7F11×CD3和7F1×CD3)对Jurkat T细胞都有较好的激活作用。
(2)检测GPC3×CD3双特异性抗体对GPC3阳性细胞的杀伤作用
所述双特异性抗体GPC3×CD3的CD3结合臂特异性地结合T细胞表面的CD3复合物，另一端GPC3结合臂特异性地结合肿瘤细胞表面GPC3分子，使T细胞与肿瘤细胞之间形成免疫桥联，继而激活T细胞，释放穿孔素(Perforin)和颗粒酶B(Granzyme B)等细胞杀伤蛋白，从而杀伤肿瘤细胞。当肿瘤细胞膜受损后，细胞膜通透性增大，胞浆中的乳酸脱氢酶(LDH)被释放到培养上清中，取一定量的上清，加入乳酸脱氢酶的反应底物乳酸后，乳酸脱氢酶催化脱氢反应，产生红色产物甲臜(Formazan)，可在490nm波长下产生吸收峰，细胞上清的乳酸脱氢酶含量越多，其颜色越深，吸光值越大，故可通过测定吸光值来定量肿瘤细胞释放乳酸脱氢酶的量，并计算GPC3×CD3介导PBMC杀伤肿瘤细胞的杀伤活性，杀伤率的计算公式为：Cytotoxicity％＝(OD 样品-SR)/(MR-SR)×100％，其中，SR＝OD 自发释放孔(靶细胞+效应细胞)，MR＝OD 最大释放孔(靶细胞)。
本公开采用GPC3高表达的细胞人肝癌细胞HepG2和Huh-7作为靶细胞，人外周血单核细胞PBMC作为效应细胞，检测所述7株GPC3×CD3双特异性抗体介导PBMC杀伤肿瘤细胞的杀伤活性。
取新鲜分离的人外周血单核细胞PBMC及长状态良好的HepG2和Huh-7细胞，分别调整细胞密度。用1×PBS缓冲液(pH 7.4)将样品稀释进行梯度稀释，取96孔细胞培养板，依次加入靶细胞、PBMC和梯度稀释的样品，混匀，使得PBMC与靶细胞的比例为20:1，把培养板置于37℃、5％CO 2培养箱内孵育，21h后，用乳酸脱氢酶细胞毒性试剂盒(碧云天)检测细胞的杀伤毒性，以抗体浓度的对数值为横坐标，杀伤活性为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC 50值，以EC 50代表GPC3×CD3对靶细胞的杀伤活性。
结果如图7A和图7B所示，结果显示，本公开构建的7株双特异性抗体GPC3×CD3对GPC3高表达的HepG2细胞有较好的杀伤效果，结果与T细胞活化结果一致，本公开构建的7株双特异性抗体GPC3×CD3对Huh-7细胞的杀伤效果稍弱于对HepG2的杀伤效果。
(3)检测GPC3×CD3双特异性抗体小鼠体内抗肿瘤作用
根据前述实施例结果，选择对T细胞激活作用及体外抗肿瘤杀伤效果较强的2株GPC3×CD3双特异性抗体7F1×CD3和7F11×CD3，进行小鼠体内抗肿瘤杀伤作用的检测。
选用7周龄的雌性BALB/c-hCD3E小鼠(购自江苏集萃药康生物科技股份有限公司)，收集对数生长期的CT26-hGPC3细胞，以2×10 6个细胞/100μL/只接种于小鼠右前肢，接种次日分组，分组当天定义为D0天，并于D0天开始给药，实验共分3组，每组6只小鼠，分别是阴性对照生理盐水组，7F1×CD3 5mg/kg组和7F11×CD3 5mg/kg组，于D0、D3、D6和D9天尾静脉注射给药(i.v.)，开始给药后，每周2次观测肿瘤大小并称量小鼠体重，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)，给药结束观察至D20，终止实验，利用肿瘤体积计算肿瘤增殖率(T/C％)和肿瘤抑制率(TGI)，根据肿瘤抑制率(TGI)进行疗效评价，肿瘤体积抑制率(TGI TV)：TGI TV(％)＝[1-(T i-T 0)/(V i-V 0)]×100％(T i：治疗组在给药第i天的肿瘤体积均值，T 0：治疗组在给药第0天的肿瘤体积均值；V i：溶剂对照组在给药第i天的肿瘤体积均值，V 0：溶剂对照组在给药第0天的肿瘤体积均值)。
结果如图8所示，7F1×CD3、7F11×CD3对均可对CT26-hGPC3移植BALB/c-hCD3E小鼠肿瘤模型具有显著抑制肿瘤生长的作用，实验终点7F1×CD3、7F11×CD3组小鼠肿瘤均消退。
综上所述，本公开提供的GPC3单克隆抗体及其亲和力成熟变体，能够以高亲和力特异性结合表达GPC3抗原的细胞，GPC3×CD3双特异抗体能够激活T淋巴细胞，有效抑制肿瘤生长并使肿瘤消退，在提高免疫细胞活性和增强免疫应答、预防治疗肿瘤和感染性疾病等方面具有重要的应用前景。
申请人声明，本公开通过上述实施例来说明本公开的详细方法，但本公开并不局限于上述详细方法，即不意味着本公开必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本公开的任何改进，对本公开产品各原料的等效替换及辅助成分的添加、方式的选择等，均落在本公开的保护范围和公开范围之内。
工业实用性
本公开提供了一种磷脂酰肌醇蛋白聚糖3抗体及其应用，本公开提供磷脂酰肌醇蛋白聚糖3抗体能够以高亲和力与磷脂酰肌醇蛋白聚糖-3特异性结合，能够激活T淋巴细胞，在提高免疫细胞活性和增强免疫应答、预防治疗肿瘤和感染性疾病等方面具有重要的应用前景，具有显著的应用性。

Claims (16)

  1. 一种磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列；
    所述磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
  2. 根据权利要求1所述的磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述磷脂酰肌醇蛋白聚糖3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列；所述磷脂酰肌醇蛋白聚糖3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
  3. 一种亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体由权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体经过突变处理制备得到。
  4. 根据权利要求3所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列。
  5. 根据权利要求3所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.14所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.20-40中任意一种所示的氨基酸序列。
  6. 根据权利要求3所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的轻链互补决定区CDR1包括SEQ ID No.17-19中任意一种所示的氨基酸序列，轻链互补决定区CDR2包括SEQ ID No.15所示的氨基酸序列，轻链互补决定区CDR3包括SEQ ID No.16所示的氨基酸序列。
  7. 根据权利要求3所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体，其特征在于，所述亲和力成熟磷脂酰肌醇蛋白聚糖3抗体的重链框架区包括如SEQ ID No.2-5所示的氨基酸序列；所述抗体的轻链框架区包括SEQ ID No.10-13所示的氨基酸序列。
  8. 生物材料，其特征在于，所述生物材料为以下B1)至B6)中的任一种：
    B1)编码(b1)或(b2)所述抗体或者(b1)或(b2)所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体，(b2)权利要求3-7任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
    B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
    B6)编码(b1)或(b2)所述抗体或者(b1)或(b2)所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。
  9. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体和/或权利要求3-7任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述药物组合物还包含一种或多种治疗剂；
    优选地，所述治疗剂包括以下的至少一种：化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂。
  11. 权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体、权利要求3-7任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或者权利要求9或10所述的药物组合物在制备治疗肿瘤药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述肿瘤包括肝癌。
  13. 权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体、权利要求3-7任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或者权利要求9或10所述的药物组合物，用于治疗肿瘤的用途。
  14. 根据权利要求13所述的用途，其特征在于，所述肿瘤包括肝癌。
  15. 一种治疗肿瘤的方法，其特征在于，所述方法包括：向所述有此需要的受试者给药权利要求1或2所述的磷脂酰肌醇蛋白聚糖3抗体、权利要求3-7任一项所述的亲和力成熟磷脂酰肌醇蛋白聚糖3抗体或者权利要求9或10所述的药物组合物。
  16. 根据权利要求15所述的方法，其特征在于，所述肿瘤包括肝癌。
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