A targeted anti-GPC3 antibody and its use

Abstract

The present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to GPC3 with higher affinity than a reference antibody and has ADCC activity.

Description

[0001] This application is a divisional application of the invention patent application with the application date of December 27, 2023; application number: 202311834579.7; the invention name is: A targeted anti-GPC3 antibody and its use. Technical Field

[0002] The present disclosure relates to the field of antibodies, and more particularly to an antibody against GPC3. Background Art

[0003] Glypican-3 (GPC3) is a heparan sulfate proteoglycan, approximately 65 kDa, anchored to the cell membrane by glycosylphosphatidylinositol. Its structure consists of a 580-amino acid core protein and two C-terminal heparan sulfate glycan side chains. GPC3 is expressed in the embryo and is virtually absent after birth, with low expression in the kidneys, gastric glands, and testes of some individuals. However, GPC3 expression is significantly elevated in over 70% of hepatocellular carcinomas. Furthermore, GPC3 is highly expressed in tumors such as ovarian and gastric cancers. The two side chains of GPC3 can regulate cell morphogenesis and growth by participating in Wnt, insulin-like growth factor, fibroblast growth factor (FGF), or Hedge-hog signaling pathways, thereby influencing tumor formation and progression. Therefore, GPC3 is a highly promising tumor-associated antigen.

[0004] Currently, GPC3-targeting therapeutics primarily focus on cell therapy and antibody-based drugs. Cell therapy drugs suffer from limited clinical benefit in solid tumors, high treatment costs, significant clinical toxicity and side effects, and a crowded competitive landscape. Clinical trials of codrituzumab, a monoclonal antibody developed by Chugai Pharmaceutical, are ongoing. Summary of the Invention

[0005] To address the aforementioned issues, the present disclosure provides antibodies, methods for preparing the same, and compositions thereof. The benefits provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnosis, and can be used in combination with antibodies reactive with various targets. The present disclosure provides antibodies that specifically bind to the GPC3 antigen, preferably chimeric or humanized monoclonal antibodies.

[0006] The present invention discloses an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to GPC3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0007] The heavy chain variable region comprises:

[0008] (i) HCDR1 comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 93-96 or consisting of SEQ ID NOs: 93-96; and

[0009] (ii) HCDR2 comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 97-106; and

[0010] (iii) HCDR3 comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 107-112;

[0011] The light chain variable region comprises:

[0012] (i) LCDR1 comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 71-78; and

[0013] (ii) LCDR2 comprising or consisting of a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity to one of SEQ ID NO: 79 (DAS), SEQ ID NO: 80 (DAF), SEQ ID NO: 81 (TAS), SEQ ID NO: 82 (LVS), and SEQ ID NO: 83 (KVS); and

[0014] (iii) LCDR3, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 84-92.

[0015] In some embodiments of the present invention, the antibody or fragment comprises the following combination:

[0016] (i) HCDR1 shown in SEQ ID NO:96, HCDR2 shown in SEQ ID NO:106, and HCDR3 shown in SEQ ID NO:112, LCDR1 shown in SEQ ID NO:78, LCDR2 shown in SEQ ID NO:83 (KVS), and LCDR3 shown in SEQ ID NO:90.

[0017] In some embodiments of the present invention, the antibody or fragment, wherein the heavy chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 54-68 or consists of one of SEQ ID NOs: 54-68.

[0018] In some embodiments of the present invention, the antibody or fragment, wherein the light chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 36-52 or consists of one of SEQ ID NOs: 36-52.

[0019] In some embodiments of the present invention, the antibody or fragment further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 70, or consisting of SEQ ID NO: 70; the light chain constant region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 69, or consisting of SEQ ID NO: 69.

[0020] In some embodiments of the present invention, the antibody or fragment comprises:

[0021] a light chain comprising or consisting of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 3-19;

[0022] A heavy chain comprising or consisting of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 20-34.

[0023] In some embodiments of the present invention, the antibody or fragment comprises:

[0024] (i) The light chain represented by SEQ ID NO: 19, and the heavy chain represented by SEQ ID NO: 34.

[0025] In some embodiments of the present invention, the antibody or fragment, wherein the antibody is selected from the following group: a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0026] In some embodiments of the present invention, the antibody or fragment, the fragment is selected from the following group: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment and ScFv.

[0027] In some embodiments of the present invention, the antibody or fragment comprises an IgG constant region, and the IgG constant region is preferably selected from IgG1, IgG2, IgG3, or IgG4 constant region, and the IgG constant region is more preferably selected from the constant region of IgG1.

[0028] In another aspect, the present invention also provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or fragment.

[0029] On the other hand, the present invention also provides a vector comprising the nucleic acid molecule.

[0030] On the other hand, the present invention also provides a host cell comprising the nucleic acid molecule or the vector.

[0031] In another aspect, the present invention also provides a conjugate comprising the antibody or fragment coupled to at least one detectable label.

[0032] In another aspect, the present invention also provides an antibody drug conjugate comprising an antibody, wherein the antibody comprises one or more drug moieties, wherein the drug moiety is covalently linked to the antibody or fragment directly or via a linker.

[0033] On the other hand, the present invention also provides a multispecific molecule comprising the antibody or antigen-binding fragment; preferably, the multispecific molecule specifically binds to GPC3 and additionally specifically binds to one or more other targets; further preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

[0034] On the other hand, the present invention also provides a pharmaceutical composition or kit, comprising the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, and a pharmaceutically acceptable carrier.

[0035] On the other hand, the present invention also provides use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a kit for diagnosing, detecting or monitoring diseases related to GPC3 expression.

[0036] On the other hand, the present invention also provides use of the antibody or fragment, or nucleic acid molecule, or vector, or host cell, or conjugate, or antibody-drug conjugate, or multispecific molecule, or pharmaceutical composition or kit in the preparation of a medicament for treating a disease associated with GPC3 expression or determining the prognosis thereof.

[0037] On the other hand, the disease associated with GPC3 expression is cancer, and the cancer is selected from the group consisting of liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, urethral cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyoma, astrocytoma, neuroblastoma, and glioma.

[0038] The present invention provides antibodies or antigen-binding fragments with novel amino acid sequences, such as mouse, human, chimeric or humanized monoclonal antibodies or antigen-binding portions thereof, which can specifically bind to human GPC3 protein with significantly higher affinity than reference antibodies currently in clinical studies.

[0039] The antibodies or antigen-binding portions thereof of the present invention have various uses, including detecting human GPC3 protein, and treating and preventing diseases associated with abnormal GPC3 expression, such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings are used to provide further understanding of the present disclosure and constitute a part of the specification. They are used to explain the present disclosure together with the embodiments of the present disclosure and do not constitute a limitation of the present disclosure.

[0041] Figure 1A-1D The results of ELISA for detecting GPC3 protein binding activity of the antibody of the present invention are shown;

[0042] Figures 2A-2D The results of flow cytometry analysis of the cell-level binding activity of the antibodies of the present invention are shown;

[0043] Figure 3A-3B The ADCC activity measurement curve of the antibody of the present invention is shown. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only illustrative embodiments of a part of the present invention, rather than all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated. In addition, any section headings used herein are not to be construed as limiting the subject matter described.

[0045] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular should include the plural, and terms in the plural should include the singular. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0046] definition

[0047] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0048] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as either kappa or lambda. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. In both light and heavy chains, the variable region is connected to the constant region by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions, CDRs for short) separated by relatively conserved regions (called framework regions, FRs for short). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus. The CDRs on VH are HCDR1, HCDR2, and HCDR3; the CDRs on VL are LCDR1, LCDR2, and LCDR3. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site / part, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of the present disclosure, the CDR sequences are determined using the numbering definitions in the IMGT system.

[0049] Antibodies in the present disclosure also include antigen-binding portions (interchangeably used with the term "antigen-binding fragment"). Antigen-binding portions refer to polypeptides comprising fragments of intact antibodies that retain the ability to specifically bind to an antigen to which the full-length or intact antibody specifically binds, and / or that compete with the full-length antibody for binding to the same antigen. Under some conditions, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and antibodies comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. The antigen-binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0050] The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0051] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / preparation of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The antibodies of the present invention can be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0052] The term "epitope" refers to an antigenic determinant in a molecule, which is a portion of a molecule that is recognized by the immune system (e.g., by an antibody), such as a discrete three-dimensional site on an antigen recognized by the immune system. In the present invention, the epitope is, for example, the GPC3 protein.

[0053] The term "chimeric antibody" as used herein refers to an antibody whose variable region sequences are from one species and the constant region sequences are from another species, for example, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0054] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In addition, additional framework region modifications may be made within the human framework sequences.

[0055] The term "KD value" refers to the equilibrium dissociation constant between an antibody and its antigen, defined as the ratio of koff / kon or kd / ka (measured using SPR technology). Therefore, the lower the KD value (the lower the concentration), the higher the antibody's affinity. Therefore, the "KD value" can be used to measure the binding affinity between an antibody and its antigen.

[0056] The terms "GPC3" and "GPC3 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human GPC3 that are naturally expressed by cells or expressed on cells transfected with the GPC3 gene. In some embodiments, binding of the disclosed antibodies to the GPC3 antigen mediates killing of GPC3-expressing cells (e.g., tumor cells) by inactivating GPC3. Killing of GPC3-expressing cells may occur through one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0057] The term "anti-GPC3 antibody" or "GPC3 antibody" refers to an antibody that is capable of binding to the GPC3 antigen or to cells expressing GPC3, as defined herein. Two types of anti-GPC3 antibodies (type I and type II anti-GPC3 antibodies) can be distinguished based on their binding properties to the GPC3 antigen and their biological activities, according to Cragg, MS et al., Blood 103 (2004) 2738-2743; and Cragg, MS et al., Blood 101 (2003) 1045-1052.

[0058] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0059] The term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide exists naturally in a living organism, and a highly pure identical polynucleotide or polypeptide separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance. For example, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0060] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be transformed, transduced, or transfected into a host cell to express the genetic material elements carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication. As for the vector for expressing the antibody, a vector type in which the antibody heavy chain and light chain are present in different vectors or a vector type in which the heavy chain and light chain are present in the same vector can be used.

[0061] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may be different from the parent cell, but are still included within the scope of the term "host cell".

[0062] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0063] The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes, but also to the ability of an organism's immune system to generate a specific immune response, such as antibodies or sensitized T lymphocytes, following antigenic stimulation. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the host's reactivity, and the immunization process.

[0064] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0065] The term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line," they also include subclones and progeny cells of the hybridoma.

[0066] The term "immune effector function" includes any function mediated by components of the immune system that results in inhibition of tumor growth and / or inhibition of tumorigenesis, including inhibition of tumor spread and metastasis. Preferably, the immune effector function results in killing tumor cells. Preferably, the immune effector function in the present invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in cells carrying tumor-associated antigens (e.g., by binding of antibodies to surface antigens) and / or inhibition of proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also simply exert their effects by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of tumor cells.

[0067] The term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis-mediated solid tumor and non-solid tumor such as leukemia that causes a medical condition. For example, cancers associated with or caused by abnormal expression of GPC3 include, but are not limited to, B-cell lymphomas, including NHL, pre-B-cell lymphocytic leukemia / lymphomas, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal, and splenic), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0068] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0069] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0070] The term "adjuvant" refers to a nonspecific immunopotentiator that can enhance the immune response to the antigen in an organism or change the type of immune response when it is delivered to the organism together with the antigen or is delivered to the organism in advance. There are multiple adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0071] Anti-GPC3 antibody

[0072] In some aspects, the invention includes isolated antibodies or antigen-binding fragments thereof.

[0073] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutants and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule, as long as it exhibits preferential association or binding with the GPC3 protein. In addition, unless the context otherwise dictates, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody, a humanized monoclonal antibody, or an improved chimeric monoclonal antibody.

[0074] The variable regions and CDRs in an antibody sequence can be identified according to the general rules (e.g., Kabat) numbering system well developed in the art, as described above, or by aligning the sequence with a database of known variable regions.

[0075] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to an antigen (e.g., GPC3) are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section regarding competition assays).

[0076] According to the present invention, an antibody is capable of binding to a predetermined target (e.g., a GPC3 protein or a cell expressing GPC3) if the antibody has significant affinity for the predetermined target in a standard assay (e.g., the assay described herein). To test the binding of a monoclonal antibody to live cells expressing GPC3, flow cytometry (FCM) can be used. Preferably, the binding of the antibody to a target expressed on the cell surface is measured in a flow cytometric fluorescence sorting (FACS) analysis. If the antibody detectably binds to the target (GPC3 protein or a cell expressing GPC3), the antibody is capable of binding to the target and has "affinity."

[0077] The GPC3 specificity of the present invention refers to the ability to bind to one or more GPC3 epitopes, especially GPC3 epitopes in their native conformation, particularly human GPC3 specificity.

[0078] In the art, various methods are used to modify antibodies without changing their desired properties, such as the recombining of light and heavy chains of antibodies and amino acid substitutions used in the present disclosure. For example, conservative amino acid substitutions can be made to the sequences of the present invention, including chimeric or humanized antibody sequences.

[0079] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences of CDRs are more diverse than other sequences between antibodies. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing an expression vector comprising CDR sequences from that specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-□327; Jones, P. et al. (1986) Nature 321:522-□525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes, which are formed by V(D)J joining during B cell maturation. The germline gene sequence will also have sequences that differ from the high-affinity second repertoire antibody at individual locations evenly across the variable region.

[0080] Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when administered repeatedly, with the primary immunogenicity mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided if the individual antibodies are chimerized or humanized.

[0081] Chimeric antibody refers to the antibody of different parts from different animal species, for example, there is the antibody of variable region and human immunoglobulin constant region from mouse antibody.The variable region of mouse antibody heavy chain and light chain is linked together with the constant region of human heavy chain and light chain to obtain the chimeric (for example, as Kraus etc., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8) of antibody.In a preferred embodiment, chimeric antibody is produced by connecting human kappa light chain constant region to mouse light chain variable region.In another preferred embodiment, chimeric antibody can be produced by connecting human lambda light chain constant region to mouse light chain variable region.

[0082] Humanized antibodies are antibodies in which CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0083] In order to reduce the immunogenicity of the antibody to humans, the sequence of the GPC3 antibody disclosed herein is used to produce a humanized anti-GPC3 antibody. The CDR region of the murine anti-GPC3 antibody is combined with a human framework region (e.g., human immunoglobulin) to form the humanized anti-GPC3 antibody of the disclosure. The humanized antibody is expected to retain the function of binding to both human GPC3 and monkey GPC3.

[0084] Preparation or production of antibodies

[0085] The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although hybridoma technology is preferred, in principle, other techniques for producing monoclonal antibodies can be used, such as viral or oncogene transformation of B lymphocytes or phage display technology using antibody gene libraries, somatic cell hybridization methods, and, for example, obtaining through genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into expression vectors, and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0086] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995), see also Rossie et al., Am. J. Clin. Pathol. 124:295 (2005)). Hybridoma production in mice is a very well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0087] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals or some combination thereof. For example, hybridomas and biochemical and genetic engineering techniques recognized in the art can be used to produce monoclonal antibodies, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed.2009; Shire et.al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed.2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0088] It will be understood that the selected binding sequence can be further altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and that antibodies comprising altered target binding sequences are also antibodies of the invention.

[0089] In some embodiments, the method of producing the antibodies or fragments described herein comprises the following steps:

[0090] (i) expressing said antibody or fragment in a host cell; and optionally

[0091] (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

[0092] In a preferred embodiment, anti-GPC3 monoclonal antibodies are produced by using hybridomas.

[0093] To obtain hybridomas that produce antibodies of the present invention, such as human monoclonal antibodies of the present invention, splenocytes and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0094] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody genes are linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody genes.

[0095] In some embodiments, the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by being inserted into the expression vector of the heavy chain constant region and the light chain constant region of the required isotype encoded, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier.In addition or alternatively, the recombinant expression vector can encode the signal peptide that promotes the secretion of antibody chain from the host cell.The antibody chain gene can be cloned into the vector so that the signal peptide is connected to the amino terminus of the antibody chain gene.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0096] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete appropriately folded and immunologically active antibodies.

[0097] Mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with a DHFR selection marker (e.g., as described in RJ Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Specifically, another expression system for use with NSO myeloma is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0098] In another preferred embodiment, transgenic or transchromosomal mice carrying parts of the human immune system (rather than the mouse system) can be used to generate human monoclonal antibodies against GPC3.

[0099] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from the antibody-producing lymphocytes of a defined strategy, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering, see also Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0100] To prepare chimeric antibodies, murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH region to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region. Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to indicate that the two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0101] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of producing a complete human antibody repertoire without endogenous immunoglobulin production upon immunization. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies upon antigenic stimulation (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and kappa light chain immunoglobulin sequences, plus targeted mutations that inactivate the endogenous μ and kappa chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or "KM mice™" carrying a human heavy chain transgene and a human light chain transchromosome (see patent application WO02 / 43478). Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0102] Nucleic acid molecules encoding the antibodies of the present invention

[0103] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above-described isolated antibody or fragment thereof as disclosed herein.

[0104] Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as further described below), cDNA encoding the light and heavy chains of the antibodies prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0105] To prepare chimeric antibodies, methods known in the art can be used to connect the mouse immunoglobulin variable region to the human immunoglobulin constant region (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). By operably connecting the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene, and DNA fragments containing these regions can be obtained by standard PCR amplification. By operably connecting the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, by converting the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0106] Conjugate

[0107] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) providing a detectable signal; (ii) interacting with a second label to modify the detectable signal provided by the first or second label, such as FRET (Fluorescence Resonance Energy Transfer); (iii) affecting mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape, or other physical parameters, or (iv) providing a capture moiety, such as affinity, antibody / antigen, or ion complexation.

[0108] Suitable structures as labels include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymer particle labels), organic small molecules (such as biotin, receptor ligands or binding molecules (such as cell adhesion proteins or lecithin), which can be used to bind to the target protein. The present invention can detect marker sequences comprising nucleic acid and / or amino acid residues by using a marker. The marker includes, but is not limited to, barium sulfate, iodine, iopanoic acid, amioprofen calcium, diatrizoate sodium, diatrizoate meglumine, meglumine, sodium tyrosine, and radiodiagnostic agents (including positron emitters (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131, and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridinium esters), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0109] Detectable labels as described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. In certain embodiments, such labels can be suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, detectable labels as described above can be connected to the antibodies or antigen-binding fragments thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0110] Antibody Drug Conjugates / Immunoconjugates

[0111] In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody, comprising one or more drug moieties / therapeutic agents, wherein the drug moiety is linked (e.g., covalently linked) directly or via a linker to an antibody or fragment thereof as described above. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-GPC3 antibody to the drug is not particularly limited, as long as the resulting antibody-drug conjugate can be used.

[0112] Because antibody-drug conjugates have the ability to selectively deliver one or more drugs to target tissues (e.g., tumor-associated antigens, such as tumors expressing GPC3), antibody-drug conjugates can improve the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0113] Multispecific molecules

[0114] The antibodies or antigen-binding fragments thereof of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present invention can be part of a multispecific molecule (e.g., bispecific molecule), and the bispecific or multispecific molecule comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, thereby being able to bind to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be connected to a second antibody or antigen-binding fragment thereof that can specifically bind to any protein that can be used as a potential target for combined therapy. To produce the bispecific or multispecific molecule, the antibodies or antigen-binding fragments thereof of the present invention can be connected (e.g., by chemical coupling, gene fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0115] Thus, in some aspects, the invention provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the invention.

[0116] In certain preferred embodiments, the multispecific molecule specifically binds GPC3 (eg, human GPC3 or monkey GPC3) and specifically binds one or more additional targets.

[0117] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0118] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0119] Pharmaceutical composition

[0120] In some aspects, the present invention relates to a pharmaceutical composition. The present disclosure provides a pharmaceutical composition or kit comprising the antibody or fragment as described above, the nucleic acid molecule as described above, the vector as described above, the host cell as described above, the conjugate as described above, the antibody-drug conjugate as described above, the multispecific molecule as described above; and a pharmaceutically acceptable carrier.

[0121] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-GPC3 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, combinations of various components known in the art, or more.

[0122] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed herein, in a solvent containing an antibody or its antigen-binding fragment of the composition disclosed herein containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment, it may be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides methods wherein antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants, such as methionine, so that the antibodies or antigen-binding fragments thereof can be protected from oxidation to extend their shelf life and / or increase their activity.

[0123] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), partitioning or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent used as a carrier can be added to the pharmaceutical composition in the multidose container comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0124] Administration, formulation and dosage

[0125] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo via various routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardial, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous formulations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Suitable formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0126] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0127] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic vehicles suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, timing, and repetition) will depend on the specific individual and individual's medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0128] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0129] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing tumor cell proliferation or delaying the development of metastases. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0130] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit with any risks or adverse side effects. The dosage level selected will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, time of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. However, the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0131] Generally, the antibodies or antigen-binding fragments thereof of the present invention can be administered in various ranges.

[0132] In certain preferred embodiments, a course of treatment involving an antibody or antigen-binding fragment thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding fragment thereof of the present invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be appreciated that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

[0133] Compatible formulations for parenteral administration (eg, intravenous injection) will contain an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL.

[0134] The anti-GPC3 antibodies of the present invention can be co-administered with one or more therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, anti-tumor agents, anti-angiogenic agents, or immunosuppressants) to reduce the induction of an immune response against the antibodies of the present invention. The antibodies can be linked to the therapeutic agent (as an immune complex) or can be administered separately from the therapeutic agent.

[0135] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which the treatments or therapeutic agents are administered to a subject. The treatment or therapeutic agent can be administered before, simultaneously with, or after the second treatment or therapeutic agent is administered to the patient. Preferably, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or at certain time intervals so that the treatment or therapeutic agent can work together. In a specific embodiment, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or at certain time intervals so that they provide an increased benefit than if administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0136] Medical uses

[0137] The antibodies, antibody compositions and methods of the present invention have many in vitro and in vivo uses, including, for example, detection of GPC3 or enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo.

[0138] Preferred subjects include mammals, such as humans / patients. Mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0139] Treating conditions associated with GPC3 expression

[0140] In some aspects, the invention provides methods of treating a disorder in a mammal comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0141] As described herein, the antibodies of the present disclosure have one or more activities that can be therapeutically used to kill cells and / or inhibit cells. In particular, killing cells, inhibiting cell proliferation and / or inhibiting cell colony formation can be used to treat or prevent cancer (including cancer metastasis). Inhibiting cell proliferation, colony formation and / or metastasis can be used, in particular, to treat or prevent cancer metastasis and the metastatic spread of cancer cells.

[0142] In some aspects, the present disclosure provides a method for treating a disease associated with GPC3 expression or determining its prognosis in a subject, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0143] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method of treating a disease associated with GPC3 expression or determining the prognosis thereof in a subject.

[0144] In some aspects, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for treating a disease associated with GPC3 expression or determining the prognosis thereof.

[0145] In one embodiment, the disease associated with GPC3 expression includes a tumor disease, such as cancer.

[0146] The antibodies or antigen-binding fragments thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiation therapy.

[0147] The antibodies or antigen-binding fragments thereof can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0148] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder, such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatics, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

[0149] diagnosis

[0150] The present invention provides methods for detecting, diagnosing or monitoring proliferative disorders in vitro and in vivo and methods for screening cells from patients to identify tumor cells including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein, and detecting the presence or absence or level of binding of the antibody to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0151] In some aspects, the present disclosure provides a method for diagnosing, detecting, or monitoring a disease associated with GPC3 expression, comprising administering an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0152] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting, or monitoring a disease associated with GPC3 expression in a subject.

[0153] In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for diagnosing, detecting, or monitoring a disease associated with GPC3 expression.

[0154] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, and the like, known to those skilled in the art.

[0155] The methods described in the present invention for detecting or monitoring GPC3 expression or the level of GPC3-expressing cells in vitro can also be used for non-diagnostic purposes.

[0156] Preferred subjects include mammals, such as humans / patients in need thereof.

[0157] The sample from the subject is blood, excreta (urine or feces), oral or nasal secretions, or alveolar lavage fluid, tissue fluid, sweat, or extracts thereof from the subject.

[0158] Drug packaging and kits

[0159] Also provided are pharmaceutical packaging and kits containing one or more containers of one or more doses of an antibody or antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose or the like; a buffer such as phosphate, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder that can be reconstituted after adding a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0160] Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and, optionally, one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy.

[0161] More specifically, the kit can have a single container containing an antibody or antigen-binding fragment thereof of the present invention, which may or may not contain additional components, or they may have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or in a manner where one component is more than another. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for holding a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0162] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container.

[0163] The present invention relates to the following sequence:

[0164] SEQ ID NO: 79CR17-62, CR17-68, CR17-77, CR17-103, CR17-152, CR17-175, CR17-177

[0165] LCDR2:DAS

[0166] SEQ ID NO:80CR17-112LCDR2:DAF

[0167] SEQ ID NO: 81CR17-124, LCDR2: TAS

[0168] SEQ ID NO: 82CR17-253, CR17-256, CR17-270LCDR2: LVS

[0169] SEQ ID NO: 83CR17-278, CR17-280, CR17-281, CR17-283, CR17-285LCDR2: KVS

[0170] Example

[0171] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise noted. The raw materials, reagents, and materials used in the following examples are commercially available products unless otherwise noted.

[0172] Example 1 Animal immunization and GPC3 antibody screening

[0173] Male Balb / c (albino) laboratory mice, MRL / MpJ (lupus erythematosus mouse model), or MRL / MpJ-Faslpr / J (lupus erythematosus with lymphoproliferation mouse), and 10-12-week-old NZW (New Zealand White) rabbits were immunized subcutaneously at multiple sites every two weeks with the full-length human GPC3 extracellular domain or the membrane-proximal peptide (P51654-1, aa 511-560) emulsified in Freund's adjuvant. Three days prior to spleen collection, selected high-titer animals were immunized with intraperitoneal injection of antigen solution or CHO-hGPC3 stably transfected cells. The harvested mouse spleen cells were then used to obtain antibody light and heavy chain variable region sequences using a single B cell screening platform. The expression vector constructed by PCR was co-transfected into 293T cells, and the cell culture supernatant was collected. Multiple positive clones that recognized the extracellular region of human GPC3 were screened by ELISA and FCM methods.

[0174] By selecting some positive clones, the light chain variable region genes were cloned into expression vectors containing the human light chain constant region to obtain light chain eukaryotic expression plasmids for the antibodies of the present invention. The heavy chain variable region genes were cloned into expression vectors containing the human IgG1 heavy chain constant region to obtain heavy chain eukaryotic expression plasmids for the antibodies of the present invention. The constructed eukaryotic expression vectors were sequenced to obtain the antibody variable region sequences shown in SEQ ID NOs: 36-68. A control antibody, GC33, was synthesized according to Nakano K et al. (2009) and expressed as a human IgG1 chimeric antibody (GC33), the sequence of which is shown in SEQ ID NOs: 1-2.

[0175] The final screening antibody was CR17-285.

[0176]

[0177] SEQ ID NO: 19CR17-285 light chain full length sequence DVLMTQTPLSLSVSLGDQASMSCRSGQSLVHSNGNTYLQWYLLKPGQSPKLLIFKVSNRFSGVPDR

[0178] FSGSGSGTDFTLKISRVEAEDLGVYFCSQTSHVPLTFGAGTKLELK

[0179] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

[0180] SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0181] SEQ ID NO: 34 CR17 - 285 full - length heavy - chain sequence QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKLTPVHGLEWIGAIEPETGGTAYNRTFK

[0182] DKATLTADKSSSTAYMELRSLTSEDSAVYYCTRYYSFAYWGQGTLVTVSS

[0183] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0184] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0185] MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

[0186] GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0187] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0188] Example 2 ELISA analysis of antibody protein level and binding activity

[0189] The purified candidate antibodies were subjected to ELISA to detect protein binding activity. The extracellular segment protein of human GPC3 antigen (without heparin sulfate glycan side chain, △HS) (Acro, GP3-H52H8-1 mg) was dispensed into a 96-well ELISA plate, 50 μl / well, and incubated at 4°C overnight; after washing 3 times with PBST, 100 μl of 3% BSA blocking solution was added and incubated at 37°C for 1 hour; after washing 3 times with PBST, 50 μl of serially diluted antibody sample (3.75 μg / ml starting concentration, 4-fold serial dilution, a total of 7 concentration points) was added and incubated at 37°C for 1 hour; after washing 3 times with PBST, 100 μl of Anti-Human IgG HRP (1:10000 dilution) was added and incubated at 37°C for 0.5 hour; after washing 4 times with PBST, 50 μl of TMB color development solution was added and color was developed at 37°C for 8 minutes, and 50 μl of ELISA stop solution was added / well to terminate the reaction. The absorbance value was measured at a wavelength of 450 nm using a microplate reader. The results are as follows: Figure 1A-1D shown.

[0190] The results showed that the candidate antibody had high binding activity to human GPC3 protein, which was basically consistent with the reference antibody.

[0191] Example 3 Flow cytometry analysis of antibody cell-level binding activity

[0192] HepG2 cells were digested with Accutase (Invitrogen, 00-4555-56) and plated at 2×10 6 The cells / ml were suspended in FACS buffer (PBS containing 3% FBS). The suspension was added to a 96-well U-bottom plate at 50 μl / well, and 50 μl of gradient diluted antibody sample (30 μg / ml starting concentration, 4-fold gradient dilution, a total of 7 concentration points) was added. The cells were incubated at 4°C for 45 minutes. The cells were washed once with FACS buffer, centrifuged, and 100 μl of FITC-labeled anti-human IgG antibody was added to each well. After incubation at 4°C for 30 minutes, the cells were washed twice and the supernatant was removed. The cells were resuspended in 100-200 μl FACS buffer and analyzed by flow cytometry. The results are shown in Figure 2. Figures 2A-2D shown.

[0193] The results showed that the candidate antibody had high binding activity to human GPC3 at the cellular level, which was better than or consistent with the reference antibody.

[0194] Example 4 Antibody Affinity Ranking

[0195] Using biolayer interferometry (BLI), the test antibody was captured on a Protein A probe and its binding to a single concentration (100 nM) of human GPC3 protein was measured using an Octet RH96 instrument. The affinity constant, KD, was calculated based on the association and dissociation rates. Affinity data are shown in Table 1:

[0196] Table 1. BLI analysis of the affinity of candidate antibodies to human GPC3 protein

[0197]

[0198] The results showed that the affinity of candidate antibodies CR17-256, CR17-270, CR17-278, CR17-283 and CR17-285 for human GPC3 protein was basically consistent with that of the reference antibody.

[0199] Example 5 Antibody Affinity Determination

[0200] Using surface plasmon resonance (SPR) technology, the test antibody was captured on a Protein A probe and its binding to human GPC3 protein at multiple concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 0 nM) was measured using a Biacore 8k instrument. The affinity constant, KD, was calculated based on the association and dissociation rates. Affinity data are shown in Table 2:

[0201] Table 2 SPR analysis of affinity of candidate antibodies to human GPC3 protein

[0202]

[0203] The results showed that the candidate antibodies CR17-256, CR17-270, CR17-278, CR17-283 and CR17-285 had better affinity for human GPC3 protein than the reference antibody.

[0204] Example 6 ADCC activity determination of candidate antibodies

[0205] Antibody ADCC activity was detected using a reporter gene assay. Jurkat-FcγRIIIa-V158 Effector Cells (from Novagen) were used. HepG2 cells (from CCTCC) grown to the logarithmic growth phase were adjusted to a cell density of 1.25E5 cells / ml using 1640+10% FBS+1% PS. The cells were then plated into a white 96-well flat-bottom plate at 100 μl / well and incubated overnight at 37°C in 5% CO2. The next day, the supernatant was removed and the volume was added to 75 μl / well. Antibody (100 nM, 5-fold serial dilutions, 7 or 8 concentration points) was added at 50 μl / well. Jurkat-FcγRIIIa-V158 Effector Cells at a cell density of 3E6 cells / ml were then added at 25 μl / well. The cells were incubated at 37°C in 5% CO2 for 6 hours. Thaw the luciferase colorimetric solution (Manufacturer: Novezan, Catalog No.: DD1204-03) 20-30 minutes in advance in room temperature water and equilibrate to room temperature. Remove the 96-well plate and equilibrate at room temperature for 5-10 minutes. Add 100 μl of the colorimetric solution per well. Incubate at room temperature in the dark for 10 minutes. Read the RLU values ​​on a multi-function microplate reader. Analyze the data using GraphPad Prism software, performing a nonlinear fit using the final antibody concentration as the horizontal axis and the RLU values ​​as the vertical axis to calculate the EC50 value.

[0206] The results are as follows Figure 3A-3B The results showed that the candidate antibodies CR17-256, CR17-270, CR17-278, CR17-283 and CR17-285 all had significant ADCC activity and could kill tumor target cells.

[0207] Incorporated by Reference

[0208] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0209] Equivalence

[0210] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to GPC3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region comprises: HCDR1 set forth in SEQ ID NO:96, HCDR2 set forth in SEQ ID NO:106, and HCDR3 set forth in SEQ ID NO:112; The light chain variable region comprises: LCDR1 represented by SEQ ID NO:78, LCDR2 represented by SEQ ID NO:83 (KVS), and LCDR3 represented by SEQ ID NO:

90.

2. The antibody or fragment of claim 1, wherein the heavy chain variable region is represented by SEQ ID NO: 68; The light chain variable region is shown in SEQ ID NO:

52.

3. The antibody or fragment according to claim 2, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is as shown in SEQ ID NO: 70; and the light chain constant region is as shown in SEQ ID NO:

69.

4. The antibody or fragment of claim 3, comprising: The light chain is represented by SEQ ID NO: 19, and the heavy chain is represented by SEQ ID NO:

34.

5. The antibody or fragment of any one of claims 1 to 4, wherein the antibody is selected from the group consisting of a whole antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

6. The antibody or fragment of any one of claims 1 to 4, wherein the fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a Fv fragment, and a ScFv.

7. The antibody or fragment of any one of claims 1 to 4, comprising the constant region of IgG.

8. The antibody or fragment according to claim 7, wherein the IgG constant region is selected from IgG1, IgG2, IgG3, or IgG4 constant region.

9. The antibody or fragment according to any one of claims 8, wherein the constant region of IgG is selected from the constant region of IgG1.

10. An isolated nucleic acid molecule encoding the antibody or fragment as claimed in any one of claims 1 to 9. A vector comprising the nucleic acid molecule according to claim 10 .

12. A host cell comprising the nucleic acid molecule according to claim 10 or the vector according to claim 11.

13. A pharmaceutical composition or kit comprising the antibody or fragment according to any one of claims 1 to 9, or the nucleic acid molecule according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, and a pharmaceutically acceptable carrier.

14. Use of the antibody or fragment according to any one of claims 1 to 9, or the nucleic acid molecule according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, or the pharmaceutical composition or kit according to claim 13 in the preparation of a kit for diagnosing, detecting or monitoring a disease associated with GPC3 expression, wherein the disease associated with GPC3 expression is cancer, and the cancer is selected from the group consisting of liver cancer, gastric cancer, and ovarian cancer.

15. Use of the antibody or fragment according to any one of claims 1 to 9, or the nucleic acid molecule according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, or the pharmaceutical composition or kit according to claim 13 in the preparation of a medicament for treating a disease associated with GPC3 expression or determining the prognosis thereof, wherein the disease associated with GPC3 expression is cancer, and the cancer is selected from the group consisting of liver cancer, gastric cancer, and ovarian cancer.

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