Phosphatidylinositol proteoglycan 3 antibodies and related methods

Abstract

The present disclosure provides amino acid sequences (e.g., antibodies) capable of binding to glypican 3 protein (GPC3). The present disclosure relates to antigen binding fragments of GPC3 antibodies; conjugates of a GPC3 antibody, including an immunoconjugate; bispecific antibodies comprising a GPC3 antibody and related compositions and formulations. The disclosure also relates to methods of treating GPC3-related diseases, disorders and conditions, including cell proliferative diseases, such as cancer, more particularly liver cancer, using these sequences, such as GPC3 antibodies and antigen-binding fragments thereof.

Description

[0001] Incorporation by reference

[0002] This application incorporates by reference the Sequence Listing submitted with this application in computer-readable form (CRF), which is an.xml file generated using WIPO Sequence Version 2.1.0, named "66348SequenceListing.xml", created on October 5, 2022, and having a size of 91,683 bytes. Background of the Invention

[0003] Liver cancer is one of the most common cancers globally. The WHO International Agency for Research on Cancer estimates that in 2018, there were over 841,000 new cases (ranking sixth) and over 781,000 deaths. Globally, Asia accounts for more than 72% of the incidence and mortality, followed by Europe, Africa, and the Americas. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two main primary liver cancers. According to the American Cancer Society, HCC accounts for approximately 75% of all liver cancer cases. The Chinese National Cancer Center estimates that HCC accounts for approximately 84% - 92% of liver cancer cases in China in 2018. Although HCC is common, treatment options remain limited in terms of the selection of approved treatment regimens, evidence from randomized controlled trials, and survival benefits.

[0004] Glypican-3 (GPC3) is highly expressed in most HCCs but not expressed in CCA and normal adult tissues. GPC3 is also expressed at low levels in other tumors. It has been reported that the 5-year survival rate of GPC3-positive HCC patients is significantly lower than that of GPC3-negative HCC patients, and GPC3 expression is associated with poor clinical prognosis of HCC.

[0005] Described herein are specific binding proteins, monoclonal antibodies, and antigen-binding fragments and domains thereof that can bind to GPC3, which can be used in methods for detecting, treating, and preventing GPC3-related disorders, diseases, and related clinical conditions (including cancer). Summary of the Invention

[0006] Generally, the present disclosure provides antibodies (i.e., isolated antibodies and antigen-binding fragments thereof) that bind to glypican-3 (GPC3). In some aspects, the present disclosure provides antibodies, isolated antibodies, and antigen-binding fragments thereof that specifically bind to epitopes of the GPC3 protein. In some embodiments, the antibody or its antigen-binding fragment may comprise one or more heavy chain variable regions, one or more light chain variable regions, one or more CDR1s of the light chain and / or heavy chain variable regions, one or more CDR2s of the light chain and / or heavy chain variable regions, one or more CDR3s of the light chain and / or heavy chain variable regions, or any combination thereof, according to the aspects and embodiments provided by the present disclosure.

[0007] In an embodiment, the antibody or antigen-binding fragment thereof binds to an epitope within the human GPC3 protein sequence (SEQ ID NO:84).

[0008] Human GPC-3 (P51654-1) (SEQ ID NO:84)

[0009]

[0010]

[0011] In some aspects, the present disclosure relates to an isolated antibody or an antigen-binding fragment thereof that binds to glypican 3 (GPC3) protein, wherein the antibody or the antigen-binding fragment thereof comprises: (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 1; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 2; (ii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 3; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 4; (iii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 5; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 6; (iv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 7; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 9; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 10; (vi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 11; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 12; (vii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 13; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14; (viii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 16; (ix) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 48;The light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:52; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:56; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xiii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:60; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xiv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:64; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:68; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xvi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:72; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xvii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:76; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; or (xviii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:80; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8.;

[0012] In some embodiments in this regard, the present disclosure provides the isolated antibody of claim 1 or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 2; (ii) a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 4; (iii) a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO: 6; (iv) a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8; (v) a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO: 10; (vi) a heavy chain variable region of SEQ ID NO: 11 and a light chain variable region of SEQ ID NO: 12; (vii) a heavy chain variable region of SEQ ID NO: 13 and a light chain variable region of SEQ ID NO: 14; (viii) a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 16; (ix) a heavy chain variable region of SEQ ID NO: 48 and a light chain variable region of SEQ ID NO: 8; (xi) a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 8; (xii) a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 8; (xiii) a heavy chain variable region of SEQ ID NO: 60 and a light chain variable region of SEQ ID NO: 8; (xiv) a heavy chain variable region of SEQ ID NO: 64 and a light chain variable region of SEQ ID NO: 8; (xv) a heavy chain variable region of SEQ ID NO: 68 and a light chain variable region of SEQ ID NO: 8; (xvi) a heavy chain variable region of SEQ ID NO: 72 and a light chain variable region of SEQ ID NO: 8; (xvii) a heavy chain variable region of SEQ ID NO: 76 and a light chain variable region of SEQ ID NO: 8; or (xviii) a heavy chain variable region of SEQ ID NO: 80 and a light chain variable region of SEQ ID NO: 8.

[0013] In some embodiments in the above aspects, the present disclosure provides an antigen-binding fragment, which comprises a single-chain Fv (scFv), a single-chain Fv-Fc (scFv-Fc), a single-chain antibody, a single-domain antibody, a Fab fragment or an F(ab')2 fragment.

[0014] In some embodiments in the above aspects, the present disclosure provides an antibody, which comprises an IgG, IgM, IgA, IgE, IgD or IgY isotype.

[0015] In some embodiments in the above aspects, the present disclosure provides a monoclonal antibody (mAb).

[0016] In some embodiments of the above aspects, the present disclosure provides an antibody or antigen-binding fragment conjugated to a structural moiety. In some embodiments, the conjugated structural moiety comprises a therapeutic agent, an active agent, a solid support, an affinity agent, or a detectable label. In some further embodiments, the structural moiety comprises a cytotoxin. In some other embodiments, the structural moiety comprises an anti-cancer agent.

[0017] In some embodiments of the above aspects, the antibody or its antigen-binding fragment binds to glypican-3 protein with a K D value of about 1.0 pM to 200 nM.

[0018] In some embodiments of the above aspects, the antibody or its antigen-binding fragment inhibits cell proliferation with an IC50 value of about 0.01 to 250 nM.

[0019] In another aspect, the present disclosure provides a bispecific antibody comprising an antibody or its antigen-binding fragment according to any of the above aspects and embodiments, and an antibody or its antigen-binding fragment that specifically binds to an antigen that does not comprise a GPC3 epitope. In some further embodiments, the bispecific antibody comprises a GPC3 antibody and a therapeutic antibody. In further embodiments, the bispecific antibody comprises an antibody or its antigen-binding fragment that specifically binds to an antigen that does not comprise a GPC3 epitope and induces an immune response. In further embodiments, the bispecific antibody comprises a GPC3 antibody or its fragment according to the present disclosure, and an antibody or its antigen-binding fragment that specifically binds to an antigen comprising CD2, CD3, CD11a, CD20, CD25 (IL2R), CD33, CD52, EGFR, VEGF, integrin α3, GPIIb / IIIar, protein F, TNF-α, TNF-β, HER2 / Neu, C5, or IgE.

[0020] In any of the above aspects and embodiments, the isolated antibody or its antigen-binding fragment may be conjugated to a solid support, an affinity agent, or a detectable agent.

[0021] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or its antigen-binding fragment according to any of the above aspects and embodiments, and a pharmaceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition or an antibody or its antigen-binding fragment according to any of the above aspects and embodiments, optional reagents, and instructions for use.

[0023] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to glypican-3 (GPC3) protein, wherein the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 1; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 2; (ii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 3; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 4; (iii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 5; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 6; (iv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 7; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 9; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 10; (vi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 11; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 12; (vii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 13; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14; (viii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15; the light chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 16; (ix) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 48;The light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:52; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:56; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xiii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:60; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xiv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:64; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xv) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:68; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xvi) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:72; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; (xvii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:76; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; or (xviii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:80; the light chain variable region comprises: CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8.;

[0024] In some further embodiments of the above aspects, the method comprises administering an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2; a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 4; a heavy chain variable region comprising SEQ ID NO: 5 and a light chain variable region comprising SEQ ID NO: 6; a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10; a heavy chain variable region comprising SEQ ID NO: 11 and a light chain variable region comprising SEQ ID NO: 12; a heavy chain variable region comprising SEQ ID NO: 13 and a light chain variable region comprising SEQ ID NO: 14; a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 16; a heavy chain variable region comprising SEQ ID NO: 48 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 52 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 56 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 60 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 64 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 68 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 72 and a light chain variable region comprising SEQ ID NO: 8; a heavy chain variable region comprising SEQ ID NO: 76 and a light chain variable region comprising SEQ ID NO: 8; or a heavy chain variable region comprising SEQ ID NO: 80 and a light chain variable region comprising SEQ ID NO: 8.

[0025] In some embodiments of the above method, wherein the cancer comprises solid tumor cell carcinoma. In some further embodiments, the cancer comprises liver cancer.

[0026] In another aspect, the present disclosure provides a method for inhibiting the proliferation of cells expressing glypican-3 (GPC3) protein, the method comprising contacting the cells with an effective amount of an antibody or an antigen-binding fragment thereof according to the above aspects and embodiments. In some embodiments of the method, the cells comprise cancer cells. In some further embodiments of the method, the cells comprise liver cancer cells.

[0027] On the other hand, the present disclosure provides a method for detecting the presence of glypican-3 (GPC3) protein in a biological sample, the method comprising contacting the biological sample with an effective amount of an antibody or an antigen-binding fragment thereof according to the above aspects and embodiments under conditions that permit the formation of a complex between the antibody or its antigen-binding fragment and GPC3 present in the sample, and measuring a detectable signal associated with the formation of the complex. In further embodiments of these methods, the antibody or its antigen-binding fragment comprises a detectable label.

[0028] In any of the above aspects and embodiments related to the method, some embodiments relate to human subjects, and / or human cells, and / or biological samples from humans.

[0029] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to the above aspects and embodiments.

[0030] In another aspect, the present disclosure provides an isolated recombinant cell that produces an antibody or an antigen-binding fragment thereof according to the above aspects and embodiments.

[0031] Based on the present disclosure and the subsequent exemplary embodiments, other aspects and embodiments of the present disclosure will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shows the cell surface binding activity of a GPC3 antibody construct according to an exemplary embodiment of the present disclosure in cells expressing GPC3 on the cell surface (HepG2) and cells not expressing GPC3 on the cell surface (NCI1975; H929).

[0033] Figure 2 shows the cellular internalization of a GPC3 antibody construct according to an exemplary embodiment of the present disclosure in HepG2 cells (2A) and Huh7 cells (2B).

[0034] Figure 3 shows the cell proliferation inhibitory activity of a GPC3 antibody construct conjugate according to an exemplary embodiment of the present disclosure in HepG2 cells (3A) and Huh7 cells (3B).

[0035] Figure 4 Shows the apoptosis activity of a GPC3 antibody construct conjugate according to an exemplary embodiment of the present disclosure in HepG2 cells.

[0036] Figure 5 Shows the calculated scores related to the heavy chain variable region sequence according to the present disclosure, determining the probability of amino acid residues interacting with the GPC3 epitope.

[0037] Figure 6 Show the binding kinetics of an affinity matured GPC3 antibody construct according to an exemplary embodiment of the present disclosure.

[0038] Figure 7 Show the ELISA curve of an affinity matured GPC3 antibody construct according to an exemplary embodiment of the present disclosure.

[0039] Figure 8 Show the cell surface binding activity of an affinity matured GPC3 antibody construct according to an exemplary embodiment of the present disclosure.

[0040] Figure 9 Show the cell surface binding activity of an affinity matured GPC3 antibody construct according to an exemplary embodiment of the present disclosure, wherein the construct has no non-specific binding activity. Detailed Description

[0041] It should be understood that the present disclosure is not limited to the specific proteins, nucleic acids, compositions, methods, or process steps described below as exemplary and illustrative aspects and embodiments, as various changes and modifications fall within the scope of this specification.

[0042] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present invention for those skilled in the art. Herein, amino acids can be represented by their commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be represented by their commonly accepted single-letter codes.

[0043] A. Antibody

[0044] As used herein, the term "antibody" or its plural "antibodies", also known as immunoglobulins, encompasses full-length antibody sequences, including, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments, bispecific antibodies, human antibodies, and humanized antibodies. As described herein, an antibody "fragment" (or "antigen-binding fragment", "binding fragment", "epitope-binding fragment", etc.) typically refers to any antibody sequence that is less than a full-length antibody sequence but still exhibits specific binding activity to a target antigen. In exemplary embodiments, an antibody fragment typically comprises a combination of at least three CDR sequences of the heavy-chain variable domain (HCDR1, HCDR2, HCDR3) and at least three CDR sequences of the light-chain variable domain (LCDR1, LCDR2, LCDR3). Some non-limiting examples of antibody fragments include single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, camelized antibodies, antibody fragments that exhibit the desired biological activity (e.g., antigen-binding portions), disulfide-linked Fv (dsFv), anti-idiotypic (anti-Id) antibodies, intrabody, and epitope-binding fragments of any of the foregoing. In some embodiments, the present disclosure provides antibodies that include an immunoglobulin molecule and an immunologically active fragment of the immunoglobulin molecule, i.e., a molecule that contains at least one antigen-binding site. Antibodies and their fragments may also include peptide fusions with the antibody or portions thereof, such as a protein fused to an Fc domain.

[0045] The immunoglobulin molecule can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), sub-isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, e.g., G1m(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)). Antibodies and their fragments can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals, such as birds (e.g., chickens).

[0046] In some aspects, the present disclosure provides novel binding agents. In some exemplary embodiments, the novel binding protein is an IgG, scFv, Fab, monoclonal antibody (mAb), or single-chain Fv-Fc (scFv-Fc) antibody. A typical or conventional mAb comprises two heavy-chain subunits and two light-chain subunits. Each mAb heavy chain comprises a variable domain (VH) involved in antigen binding and consists of three or four subregions (C H 1, C H2. C H 3. C H 4) The constant domains (CH) that make up. VH contains three complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Each mAb light chain contains a variable domain (VL) and a constant domain (CL). VL contains three CDRs: LCDR1, LCDR2, and LCDR3. Two light chain constant domain isotypes are found in mammals: κ (kappa) and λ (lambda). Through disulfide bonds, each C H 1 domain is linked to a CL domain, and the two C H 2 domains are linked to each other. Five types of heavy chains (α, δ, ε, γ, and μ) are found in different types of antibodies (IgA, IgD, IgE, IgG, and IgM). The mAb heavy chain has a hinge region that confers structural flexibility and mobility.

[0047] The "Fc" region encompasses domains from the constant region of the immunoglobulin heavy chain, including its fragments, analogs, variants, mutants, or derivatives. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other types such as IgA, IgD, IgE, and IgM. The Fc region can be a native sequence Fc region or an altered Fc region. The Fc region of an immunoglobulin typically contains two constant domains, namely C H 2 domain and C H 3 domain. The "Fv" region contains the VH and VL domains of the immunoglobulin. The "scFv-Fc" antibody used herein refers to a fusion protein of a single VH domain and a single VL domain linked by a hinge region to the C H 2 domain and C H 3 domain.

[0048] As described herein, the antibodies and antigen-binding fragments thereof disclosed herein contain binding domains that bind to GPC3 epitopes. The "binding domain" or "binding sequence" can be used interchangeably with an antibody fragment or "antigen-binding fragment" and refers herein to the portion of the antibody sequence that is sufficient to bind to a target structure, antigen, or epitope, or to interact with a target structure, antigen, or epitope. In some aspects, the antibody binds to a region of GPC3 that contains the antigenic sequence or fragment of SEQ ID NO:84. In other aspects, the antibody binds to a domain of the GPC3 protein. For example, in some embodiments, the binding domain can specifically bind to an epitope in the N-terminal subunit of GPC3 with a molecular weight of approximately 40 kDa. In some embodiments, the binding domain can specifically bind to an epitope in the C-terminal subunit of GPC3 with a molecular weight of approximately 30 kDa.

[0049] In some embodiments, the antibodies and antigen-binding fragments thereof according to the present disclosure bind to GPC3 and can inhibit the binding interaction of GPC3 with other molecules. In some exemplary embodiments, the antibodies and antigen-binding fragments thereof can inhibit or antagonize one or more GPC3-related signaling pathways. Non-limiting examples of such activities include cellular pathways related to cell division, cell growth regulation, Wnt / β-catenin signaling, frizzled signaling, protocadherin FAT1 signaling, or Yap signaling. According to such exemplary embodiments of the present disclosure, the antibody or its fragment can prevent, inhibit, or reduce the binding and / or activity of GPC3 and can provide neutralizing activity against GPC3 and its downstream signaling partners and pathways.

[0050] In some embodiments, the antibodies disclosed herein are characterized by one or more of the following structural and / or functional properties:

[0051] a. comprising amino acid sequences of a VH domain and a VL domain, wherein the VH domain comprises HCDR1, HCDR2, and HCDR3 determined in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 48, 52, 56, 60, 64, 68, 72, 76, or 80; and wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 determined in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16;

[0052] b. an amino acid sequence comprising a VH domain having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 48, 52, 56, 60, 64, 68, 72, 76, or 80 and / or a VL domain having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16;

[0053] c. An amino acid sequence having: an HCDR1 comprising SEQ ID NO: 85, 86, 97, 49, 53, 57, 61, 65, 69, 73, 77 or 81; an HCDR2 comprising SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78, 82, 87, 88 or 98; an HCDR3 comprising SEQ ID NO: 51, 55, 59, 63, 67, 71, 75, 79, 83, 89, 90, 91, 99, 100 or 101; and / or an LCDR1 comprising SEQ ID NO: 93; an LCDR2 comprising SEQ ID NO: 94; an LCDR2 comprising SEQ ID NO: 95 or 96;

[0054] d. Binding specificity for GPC3;

[0055] e. For GPC3, the dissociation constant (K D ) is in the range of 1 pM - 500 nM, 10 pM - 500 nM, 0.1 nM - 500 nM, 1 - 450 nM, 1 - 400 nM, 1 - 300 nM, 1 - 200 nM, 1 - 100 nM, 1 - 50 nM, 1 - 25 nM or 1 - 10 nM;

[0056] f. For GPC3, the IC50 value is in the range of 0.01 - 250 nM, 0.01 - 200 nM, 0.01 - 150 nM, 0.01 - 100 nM, 0.01 - 50 nM, 0.01 - 25 nM, 0.01 - 20 nM or 0.01 - 10 nM;

[0057] g. Capable of inhibiting, reducing, preventing or disrupting GPC3 binding interactions, as measurable by assay methods including, for example, filter - binding assay, fluorescence spectroscopy, ELISA, isothermal titration calorimetry and surface plasmon resonance.

[0058] In some aspects, an antibody or an antigen - binding fragment thereof that is capable of inhibiting, reducing, preventing or disrupting the binding activity or biological activity of GPC3 and / or capable of neutralizing GPC3 activity binds to an epitope present in the GPC3 protein. In some exemplary embodiments, the epitope to which the antibody or its fragment binds may be in the C - terminal domain. In some other embodiments, the epitope to which the antibody or its fragment binds may be in the N - terminal domain.

[0059] In some aspects, an antibody or an antigen - binding fragment thereof that binds GPC3 comprises a VH domain having an amino acid sequence of SEQ ID NO: 48, 52, 56, 60, 64, 68, 72, 76 or 80.

[0060] In some aspects, an antibody or an antigen-binding fragment thereof that binds to GPC3 comprises a VL domain having the amino acid sequence of SEQ ID NO:8.

[0061] In some embodiments of these aspects, an antibody or an antigen-binding fragment thereof that binds to GPC3 comprises a combination of VH and VL domains selected from:

[0062] a heavy chain variable region comprising SEQ ID NO:1; and a light chain variable region comprising SEQ ID NO:2;

[0063] a heavy chain variable region comprising SEQ ID NO:3; and a light chain variable region comprising SEQ ID NO:4;

[0064] a heavy chain variable region comprising SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:6;

[0065] a heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:8;

[0066] a heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:10;

[0067] a heavy chain variable region comprising SEQ ID NO:11; and a light chain variable region comprising SEQ ID NO:12;

[0068] a heavy chain variable region comprising SEQ ID NO:13; and a light chain variable region comprising SEQ ID NO:14;

[0069] a heavy chain variable region comprising SEQ ID NO:15; and a light chain variable region comprising SEQ ID NO:16;

[0070] a heavy chain variable region comprising SEQ ID NO:48; and a light chain variable region comprising SEQ ID NO:8;

[0071] a heavy chain variable region comprising SEQ ID NO:52; and a light chain variable region comprising SEQ ID NO:8;

[0072] a heavy chain variable region comprising SEQ ID NO:56; and a light chain variable region comprising SEQ ID NO:8;

[0073] a heavy chain variable region comprising SEQ ID NO:60; and a light chain variable region comprising SEQ ID NO:8;

[0074] comprising a heavy chain variable region of SEQ ID NO:64; and a light chain variable region of SEQ ID NO:8;

[0075] comprising a heavy chain variable region of SEQ ID NO:68; and a light chain variable region of SEQ ID NO:8;

[0076] comprising a heavy chain variable region of SEQ ID NO:72; and a light chain variable region of SEQ ID NO:8;

[0077] comprising a heavy chain variable region of SEQ ID NO:76; and a light chain variable region of SEQ ID NO:8; or

[0078] comprising a heavy chain variable region of SEQ ID NO:80; and a light chain variable region of SEQ ID NO:8.

[0079] In certain embodiments in these respects, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain having a combination of heavy and light chains or a combination of heavy and light chain CDR sequences as shown in Table 1.

[0080] In exemplary embodiments, the antibodies disclosed herein exhibit binding specificity for the GPC3 protein and can inhibit, reduce, prevent, or disrupt GPC3 binding interactions.

[0081] Those skilled in the art will understand that the sequences disclosed herein can be modified to a certain extent without impairing the ability of the antibody or its fragment to interact with the target protein (i.e., GPC3). In certain aspects, the antibody sequence variants retain the ability to interact with GPC3, thereby disrupting, preventing, reducing, or inhibiting the binding interaction between GPC3 and any one or more of its binding partners.

[0082] As used herein, the term "variant" of a sequence refers to an antibody amino acid sequence comprising at least one amino acid insertion, deletion, and / or substitution, wherein the resulting antibody retains one or more of the antibody functional characteristics described herein. In some embodiments, the sequence variant retains the ability to specifically bind the GPC3 protein. Amino acid insertion variants are characterized by the insertion of one or more amino acids between two existing amino acids. Amino acid deletion variants are characterized by the deletion of one or more amino acids from the antibody sequence. Amino acid substitutions are characterized by the replacement of at least one amino acid in the sequence with another amino acid. In embodiments involving substitutions, the (one or more) amino acid substitutions can be conservative substitutions (i.e., an amino acid from one amino acid family (based on side chain characteristics, including size, which can be acidic, basic, nonpolar, and uncharged) is replaced with an amino acid from the same family).

[0083] In some aspects, the sequence identity between the variant antibody sequence and the antibody sequences disclosed herein will be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. "Sequence identity" refers to the percentage of amino acid residues that are identical to the sequence being compared.

[0084] B. Labels, conjugates, and structural moieties

[0085] The binding proteins, antibodies, or antigen-binding fragments thereof disclosed herein can be conjugated to a therapeutic agent, a solid support, an affinity agent, or a detectable agent. For example, the amino acid sequences disclosed herein can be conjugated to a label for diagnostic purposes and other assay tests for detecting the amino acid sequence (i.e., the antibody and / or its associated target). Labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles (microparticles and nanoparticles of various materials (polymers, magnetic, etc.)), haptens, enzymes, peptides, radioisotopes, etc., and combinations thereof.

[0086] In certain aspects, the antibody is conjugated to a fluorophore. The choice of fluorophore attached to the antibody will determine the absorption and fluorescence emission characteristics of the conjugated antibody. Physical properties of fluorophore labels that can be used for antibodies and antibody-binding ligands include, but are not limited to, spectral properties (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, or combinations thereof. All of these physical properties can be used to distinguish one fluorophore from another, thus allowing for multiplex analysis. Other desirable properties of the fluorescent label can include cell permeability and low toxicity (e.g., if antibody labeling is to be performed in cells or organisms (e.g., live animals)).

[0087] In some aspects, the conjugated label can include an enzyme. In some embodiments, the enzyme is a desirable label because amplification of the detectable signal can be obtained and increased assay sensitivity results. The enzyme itself does not produce a detectable reaction, but when it is contacted with a suitable substrate, it functions to break down the substrate such that the converted substrate produces a fluorescent, colorimetric, or luminescent signal. The enzyme can amplify the detectable signal because one enzyme on the labeling reagent can cause multiple substrates to be converted into detectable signals. Enzyme substrates are selected to produce a preferred measurable product, such as a colorimetric, fluorescent, or chemiluminescent product. Such substrates are widely used in the art and are well known to those skilled in the art, including, for example, oxidoreductases (such as horseradish peroxidase) and substrates (such as 3,3'-diaminobenzidine (DAB)); phosphatases such as acid phosphatase, alkaline phosphatase, and substrates such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, such as β-galactosidase, β-glucuronidase, or β-glucosidase, and substrates such as 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal); other enzymes, including hydrolases such as cholinesterase and peptidases, oxidases such as glucose oxidase and cytochrome oxidase, and reductases for which suitable substrates are known.

[0088] Enzymes that produce chemiluminescence and their suitable substrates are useful for certain assay tests. These include, but are not limited to, luciferase and aequorin in their native and recombinant forms. In addition, chemiluminescent substrates for phosphatases, glycosidases, and oxidases (such as substrates containing stable dioxetanes, luminol, isoluminol, and acridinium esters) are also useful.

[0089] On the other hand, haptens, such as biotin, can also be used as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal and can be used as a tag for affinity chromatography separation purposes. For detection purposes, an enzyme conjugate that has an affinity for biotin, such as avidin-HRP, can be used. A peroxidase substrate is then added to produce a detectable signal.

[0090] Haptens also include hormones, natural and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and the like.

[0091] In some aspects, fluorescent proteins can be conjugated to antibodies as labels. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliprotein and their derivatives. Fluorescent proteins, particularly phycobiliproteins, are especially useful for constructing tandem dye-labeled labeling reagents. These tandem dyes contain a fluorescent protein and a fluorophore and are used to obtain a larger Stokes shift, where the emission spectrum is more shifted from the wavelength of the absorption spectrum of the fluorescent protein.

[0092] In some aspects, the label is a radioisotope. Examples of suitable radioactive materials include, but are not limited to, iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113 mIn, 115 mIn,), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 135 Xe), fluorine ( 18 F), 153 SM, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh and 97 Ru.

[0093] In some aspects, a drug can be conjugated to an antibody. For example, an antibody can be conjugated to a therapeutic moiety or agent, such as an anti-proliferative drug, an anti-cancer drug, a cytotoxic drug, or an antiviral drug. In some embodiments, the antibody can bind to a target antigen, and the drug (e.g., an anti-cancer drug) can inhibit, inactivate, or kill cancer cells. Thus, any drug known in the art can be conjugated to an antibody or a fragment thereof according to the present disclosure. In certain embodiments, the drug and other molecules can be conjugated to the antibody in a site-specific conjugation manner.

[0094] C. Polynucleotides Encoding Antibodies and Recombinant Cells Producing Antibodies

[0095] The present disclosure provides methods for generating antibodies and antibody fragments. In certain aspects, the present disclosure provides recombinant methods for generating antibodies and / or their fragments. In some embodiments, a recombinant nucleic acid encoding an antibody or a fragment thereof can be operably linked to one or more regulatory nucleotide sequences in an expression construct. In some embodiments, the nucleic acid sequences encoding the light and heavy chains of an antibody can be cloned in the same expression vector in either orientation (e.g., the light chain before the heavy chain or vice versa), or can be cloned in two different vectors. If expression is carried out using one vector, the two coding genes can have their respective genetic elements (e.g., promoter, RBS, leader sequence, termination sequence, polyA, etc.), or they can be cloned to have a single set of genetic elements but be linked by a cis - acting element. The regulatory nucleotide sequences can be suitable for the host cell used for expression. A variety of types of suitable expression vectors and suitable regulatory sequences known in the art are available for various host cells. Typically, one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome - binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Any known constitutive or inducible promoter can be considered for the aspects and embodiments covered by the present disclosure. The promoter can be a naturally occurring promoter, or can be a hybrid promoter that combines elements of multiple promoters. The expression construct can be present on an episome (e.g., a plasmid) in the cell, or the expression construct can be inserted into the chromosome.

[0096] In some aspects, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are known and can vary depending on the host cell used. In some aspects, the present disclosure relates to an expression vector that contains a nucleotide sequence encoding a polypeptide, which is operably linked to at least one regulatory sequence. Regulatory sequences are generally known and can be selected to direct the expression of the encoded polypeptide. Thus, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements. Exemplary non-limiting regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector can depend on factors such as the choice of host cell to be transformed and / or the type of protein to be expressed (e.g., an antibody or a fragment thereof). In addition, the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) can also be considered.

[0097] In some embodiments related to methods for producing antibodies, host cells can be transfected with one or more expression vectors encoding an antibody or a fragment thereof (e.g., a single vector encoding the heavy and light chains; or two vectors, one encoding the heavy chain and one encoding the light chain) and cultured under appropriate conditions to allow polypeptide expression. The antibody or a fragment thereof can be secreted and isolated from the cells and / or cell culture medium containing the antibody or a fragment thereof. Alternatively, the antibody can be retained in the cytoplasmic or membrane fraction, the harvested cells can be lysed, and subsequently purified and isolated. Cell culture includes host cells, culture medium, and other by-products. Any suitable cell culture medium can be used for the production method. Conventional techniques for purifying proteins, particularly antibodies, including, for example, ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification, can be used to isolate antibodies and antibody fragments from the cell culture medium, host cells, or both. In some aspects, the antibody can be produced as a fusion protein that contains a domain that facilitates its purification (e.g., a His tag).

[0098] Recombinant nucleic acids can be produced by ligating a cloned gene or a portion thereof to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vectors for the production of recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids, for expression in prokaryotic cells (e.g., Escherichia coli). In some aspects, mammalian expression vectors contain both prokaryotic sequences (to facilitate vector amplification in bacteria) and one or more eukaryotic transcription units for expression in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids (e.g., pBR322) to facilitate their replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, virus derivatives, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient expression of proteins in eukaryotic cells. A variety of methods for preparing plasmids and transforming host organisms are well known in the art. For other expression systems suitable for prokaryotic and eukaryotic cells and general recombinant procedures, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989). In some cases, it may be desirable to use a baculovirus expression system to express recombinant polypeptides. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., pBlueBac III containing β-gal).

[0099] Techniques for preparing fusion genes are well known. Basically, the ligation of various nucleic acid fragments encoding different polypeptide sequences can be carried out according to conventional techniques, using blunt ends or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as needed, treatment with alkaline phosphatase to avoid unwanted ligation, and enzymatic ligation. On the other hand, fusion genes can be synthesized by conventional techniques, including using an automated DNA synthesizer. Alternatively, PCR amplification of gene fragments can be carried out using anchored primers that generate complementary overhangs between two consecutive nucleic acid fragments, which can then be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons: 1992).

[0100] In some aspects, an expression vector expressing any of the above nucleic acids can be used to express an antibody in a host cell. For example, an antibody can be expressed in a bacterial cell (e.g., Escherichia coli), an insect cell (e.g., using a baculovirus expression system), yeast, or a mammalian cell. Other suitable host cells are known to those skilled in the art.

[0101] Once the expression vector has been transferred into a host cell by conventional techniques, the transfected cells can be cultured by conventional techniques to produce an antibody. Accordingly, the present disclosure includes host cells containing a polynucleotide encoding an antibody or an antigen-binding fragment thereof, which polynucleotide is operably linked to a heterologous promoter. In certain aspects, the heavy and light chains can be co-expressed in a host cell (from the same or different vectors) to express the whole antibody. In certain aspects, both the heavy and light chains of the antibody are expressed from a single promoter. In certain aspects, the heavy and light chains of the antibody are expressed from multiple promoters. In certain aspects, the heavy and light chains of the antibody are encoded on a single vector. In certain aspects, the heavy and light chains of the antibody are encoded on multiple vectors.

[0102] Mammalian cell lines that can be used as hosts for recombinant antibody expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and many other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. A suitable cell line or host system can be selected to ensure proper modification and processing of the expressed antibody or its portions. For this purpose, eukaryotic host cells with the cellular machinery for proper processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include but are not limited to CHO, HEK293, VERO, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells.

[0103] In some aspects, the antibodies and antibody fragments of the present disclosure are stably expressed in cell lines. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, a cell line that stably expresses an antibody molecule can be generated. A host cell can be transformed with a suitable engineered vector containing expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. After introduction of the foreign DNA, the cells can be allowed to grow in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells in which the plasmid has been stably integrated into their chromosomes to grow and form colonies, and the cells can then be cloned and expanded into a cell line. Methods for producing high-yield stable cell lines are well known in the art and the reagents are generally commercially available.

[0104] In some aspects, the antibodies and antibody fragments of the present disclosure are transiently expressed in cell lines. Transient transfection refers to the process in which nucleic acids introduced into a cell are not integrated into the genome or chromosomal DNA of the cell, but are maintained in the cell as extrachromosomal elements (e.g., as episomes). The transcription process of episomal nucleic acids is not affected, and proteins encoded by the nucleic acids of the episomes are produced.

[0105] Stably or transiently transfected cell lines can be maintained in cell culture media and conditions well known in the art, resulting in the expression and production of monoclonal antibodies. In some aspects, mammalian cell culture media are based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other aspects, the cell culture media are modified to support increased cell growth and biologic protein expression both. As used herein, the terms "cell culture media", "media", and "media formulation" refer to nutritive solutions used to maintain, grow, proliferate, or expand cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for specific cell culture uses, including, for example, cell culture growth media formulated to promote cell growth, or cell culture production media formulated to promote recombinant protein production. The terms "nutrients", "ingredients", and "components" are used interchangeably herein to refer to the constituents that make up cell culture media.

[0106] Once produced, antibodies can be purified by any method known in the art for purifying immunoglobulin molecules or other multimeric molecules, e.g., by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography on specific antigen Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins.

[0107] When using recombinant techniques, antibodies and antibody fragments can be produced intracellularly, in the periplasmic space, or secreted directly into the culture media. If the molecule is produced intracellularly, in a first step, particulate debris (host cells or lysed fragments) is removed by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology, 10:163-167 (1992) describe a method for isolating antibodies where the antibody is secreted into the periplasmic space of E. coli. When antibody molecules are secreted into the culture media, the supernatant from the expression system is typically first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors (e.g., PMSF) can be added at any of the above steps to inhibit proteolysis, and antibiotics can be added to prevent the growth of adventitious contaminants.

[0108] Compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography, either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain (if present) in the molecule, and this is understood by those skilled in the art. The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices can also be used. Mechanically stable matrices, such as controlled pore glass or poly(styrene divinylbenzene), can achieve faster flow rates and shorter processing times than agarose. Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin chromatography, SEPHAROSE chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, can also be used depending on the molecule to be recovered.

[0109] Regardless of how the antibody or antibody fragment is purified, in order to confirm functional binding activity, a binding assay can be performed (before and / or after purification). For example, an ELISA assay can be used, including a double ELISA assay. In some aspects, a first antigen is coated on the wells and the antibody is immobilized by binding to the antigen. A labeled second antigen is added to the wells and detected. Only the antibody that is immobilized by binding to the first antigen and binds to the second antigen will be detected. In some aspects, the present disclosure provides recombinant cell lines that can be deposited and stored in an international depository authority authorized under the Budapest Treaty (i.e., the International Depository Authority, IDA).

[0110] D. Pharmaceutical Formulations

[0111] In certain aspects, the present disclosure provides pharmaceutical compositions. The pharmaceutical composition can also be a composition comprising an antibody and / or antibody fragment disclosed herein and a pharmaceutically acceptable excipient. In certain aspects, the pharmaceutical compositions of the present disclosure are used as a medicament (i.e., for treating or preventing a disease or disorder (e.g., cancer or its clinically relevant symptoms) in a subject in need of treatment or prevention). In some embodiments, the pharmaceutical composition can be a composition comprising a nucleic acid molecule encoding an antibody disclosed herein.

[0112] In certain aspects, an antibody (or a nucleic acid molecule encoding the antibody) can be formulated with a pharmaceutically acceptable carrier, excipient, or stabilizer into a pharmaceutical composition. In certain aspects, the pharmaceutical composition is suitable for administration to a human or non-human mammal or animal via one or more routes of administration using methods known in the art. The route of administration and / or mode will vary depending on the desired outcome. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations typically may contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for human administration. Other carriers, excipients, and / or additives that may be considered for the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweetening agents, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), and the like. These and other known pharmaceutical carriers, excipients, and / or additives suitable for the formulations described herein are known in the art and are listed, for example, in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005), and "Physician’s Desk Reference", 60th ed., Medical Economics, Montvale, N.J. (2005). The pharmaceutically acceptable carrier can be selected to be suitable for the desired or required mode of administration, solubility, and / or stability.

[0113] The formulations described herein contain an active agent (i.e., one or more of the antibodies or fragments thereof described herein) at a concentration such that its weight / volume ratio (w / v) is suitable for the desired dose. In certain aspects, the active agent is present in the formulation at a concentration of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, or about 1 mg / ml to about 25 mg / ml. In certain aspects, the concentration of the active agent in the formulation can be about 0.1% to about 75% of the total weight. In certain aspects, the concentration of the active agent ranges from 0.003 to 1.0 mole.

[0114] When used for in vivo administration, the formulation should be sterile. The formulation can be sterilized by various sterilization methods, including sterile filtration, radiation, etc. On the one hand, the formulation is filtered and sterilized using a pre-sterilized 0.22-micron filter. The sterile injectable composition can be formulated according to conventional pharmaceutical practices as described in “Remington: The Science & Practice of Pharmacy”, 21st Edition, Lippincott Williams & Wilkins, (2005).

[0115] Accordingly, in embodiments, the therapeutic composition is encompassed in the pharmaceutical formulations described herein and can be formulated for a particular route of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0116] The formulation can be in unit dosage form and can be prepared by any known method. The actual dosage level of the active ingredient in the pharmaceutical composition can vary to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient (e.g., a “therapeutically effective amount”). The selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and other factors well known in the medical arts. Suitable dosage ranges can be from about 0.0001 to about 100 mg / kg body weight or higher, such as about 0.1, 1, 10, or 50 mg / kg body weight, with about 1 to about 10 mg / kg body weight being suitable.

[0117] In some embodiments of the present disclosure, the formulation can be suitable for diagnostic and research uses. The concentration of the active agent in such formulations, as well as the presence or absence of excipients and / or pyrogens, can be adjusted or selected according to the specific application and intended use.

[0118] E. Methods

[0119] The antibodies and fragments thereof described herein can be used in methods for blocking, inhibiting, or reducing the activity and / or expression of GPC3 in a subject. In this regard, the antibodies and fragments thereof disclosed herein can provide neutralizing effects against GPC3. In some aspects, the antibodies or fragments of the present disclosure can be used to treat GPC3 disorders, diseases, or conditions in a subject in need of treatment. In some aspects, the antibodies and fragments disclosed herein can be used to prevent GPC3-related disorders, diseases, or conditions in a subject who may be at risk of developing the disorder, disease, or condition. In some aspects, the antibodies can be used to treat cancers associated with GPC3 activity and / or expression in a subject in need of treatment.

[0120] In some aspects, the present disclosure relates to methods for treating, preventing, diagnosing, or monitoring disorders, diseases, or conditions characterized by GPC3, and in some embodiments, the disease is cancer. In these aspects, the antibodies, compositions, and methods described herein can be used to treat subjects suffering from GPC3-related disorders, diseases, or conditions, such as cancers characterized by GPC3 expression and / or activity.

[0121] In an embodiment, the present disclosure provides a treatment method that includes an antibody or an antigen-binding fragment thereof according to the present disclosure, which can provide effective treatment or "disease control" (DC). Disease control can be complete response (CR), partial response (PR), or stable disease (SD).

[0122] "Complete response" (CR) means that all tumors or lesions (whether measurable or not) disappear, and no new tumors or lesions form. It can be confirmed by repeated, consecutive evaluations (e.g., for at least a specified number of weeks (e.g., 4 weeks) since the first record date). New, non-measurable tumors or lesions are excluded from CR.

[0123] "Partial response" (PR) means that the tumor burden is reduced by ≥50% relative to the baseline. It can be confirmed by consecutive repeated evaluations (e.g., for at least a specified number of weeks (e.g., 4 weeks) since the first record date).

[0124] "Disease progression" (PD) means that the tumor burden increases by ≥25% relative to the lowest recorded value (nadir). It can be confirmed by consecutive repeated evaluations (e.g., for at least a specified number of weeks (e.g., 4 weeks) since the first record date). New, non-measurable lesions do not define PD.

[0125] "Stable disease" (SD) means that the criteria for CR, PR, or PD are not met. SD indicates that it cannot be established that the tumor burden is reduced by 50% relative to the baseline, nor can it be established that the tumor burden increases by 25% relative to the nadir.

[0126] "Disease" refers to any illness or disorder that impairs or interferes with the normal function of cells, tissues, or organs. In the aspects and embodiments disclosed herein, the disease is typically cancer, such as solid tumors. In some embodiments, the cancer may include liver cancer. Liver cancer includes but is not limited to hepatocellular carcinoma (HCC), cholangiocarcinoma, intrahepatic carcinoma (IHC), or angiosarcoma. Signs associated with the development of liver cancer may include cirrhosis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic inflammation (such as inflammation associated with bile ducts), and increased GPC3 expression or activity (as discussed herein). Although early-stage liver cancer may not have any associated symptoms or clinical signs, typical clinical symptoms associated with liver cancer (such as HCC and IHC) may include a mass under the rib cage, pain in the right abdomen or shoulder, jaundice, weight loss, nausea, loss of appetite, fatigue, and dark urine, etc.

[0127] Liver cancer can be diagnosed by detection methods well-known in the art, such as by blood screening to detect liver function, liver enzymes, proteins (alpha-fetoprotein (AFP)), and other analytes indicating general liver health, as well as ultrasound, CT scan, MRI, angiography, liver biopsy, endoscopic retrograde cholangiopancreatography (ERCP), and percutaneous transhepatic cholangiography (PTC), etc. In some embodiments of the present disclosure, the expression and / or activity of GPC3 can be used to evaluate the likelihood that a subject has or may develop a certain type of liver cancer.

[0128] In some embodiments of various aspects, the present disclosure relates to determining the expression or activity of GPC3 in a sample obtained from a subject who may have cancer (such as, solid tumor cancer, such as liver cancer), which can be used to identify patients who may respond to the antibody therapy of the present disclosure. In some embodiments, the determination may include detecting the mRNA encoding GPC3 and can be used to identify patients who may respond to GPC3 antibody therapy. In some embodiments, the methods disclosed herein include detecting the amount and / or activity of GPC3 protein to determine the responsiveness of cancer to GPC3 antibody therapy. In some further embodiments, the methods disclosed herein include detecting the amount and / or activity of GPC3 protein by contacting the sample (i.e., patient sample) with the antibody of the present disclosure or its antigen-binding fragment, and detecting the presence in the sample (such as, the amount and / or activity of GPC3). In such embodiments, the method can be used to evaluate or determine the responsiveness of cancer to GPC3 antibody therapy. The methods disclosed herein may include analyzing blood samples, biopsy samples, or immunohistochemical techniques applied to individual tissue and / or tumor samples.

[0129] In some of the above aspects and embodiments, the present disclosure provides methods that include detecting GPC3, thereby identifying cancers in a subject that are likely to exhibit a positive clinical response before or during treatment, and also allowing modification of the treatment based on the method when no response or a low likelihood of response to the current therapy is likely to be observed or expected. In some embodiments, the GPC3 antibodies or antigen-binding fragments thereof of the present disclosure can be used in the detection method.

[0130] In one aspect, the present disclosure provides a method of treating a subject's cancer, including administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, wherein it is determined that the subject is to receive treatment by detecting an increase in the level of GPC3 in a sample obtained from the subject. In some embodiments of this aspect, the present disclosure provides a method of treating a subject's liver cancer.

[0131] As used herein, "treatment" refers to administering to a subject an antibody or antigen-binding fragment thereof or a composition described herein to eliminate or alleviate the clinical signs of a disorder, disease, or condition; prevent, inhibit, or slow the progression of a disorder, disease, or condition in a subject; and / or reduce the number, frequency, or severity of the clinical symptoms and / or recurrence of a disorder, disease, or condition in a subject currently suffering from or previously suffering from a disorder, disease, or condition (e.g., cancer and / or cancer recurrence). In particular, the term "treatment of a disease" (e.g., a disease related to cell proliferation, cancer, etc.) includes causing a disease or its symptoms to stop, improve, alleviate, shorten its duration, slow or inhibit its progression or worsening, or delay its onset in a subject suffering from the disease.

[0132] In some embodiments, the present disclosure provides methods for preventing a disease in a subject (i.e., preventing a disorder, disease, or condition, preventing the onset of a disorder, disease, or condition, or the clinical symptoms of a disease). In some further embodiments of the methods related to preventing a disease, the subject may be at risk of one or more GPC3 disorders, diseases, or conditions. "At risk" or having an "increased risk" means that the subject is identified as having a higher than normal probability of developing a disorder, disease, or condition and / or its related clinical symptoms compared to the general population. In some embodiments, a subject who has or currently has a disorder, disease, or condition, or is of a particular age is a subject at increased risk of developing a disorder, disease, or condition. In some aspects, a subject exhibiting increased GPC3 expression or activity can also be considered at increased risk of developing a disorder, disease, or condition. In some embodiments, the subject can also be immunocompromised. As used herein, "immunocompromised" means that the subject has a weakened immune system or a decreased ability to resist infection or other diseases due to a genetic disorder or disease, an infection, an environmental disorder or disease, or other environmental factors.

[0133] The term "immunotherapy" refers to treatments involving specific immune responses. In the context of the present invention, terms such as "protection", "prevention", "preventive" or "protective" refer to preventing the occurrence and / or spread of a disorder, disease or condition and clinical symptoms in an individual, and in some embodiments, to minimizing the likelihood of further clinical symptoms or disease progression in a subject. For example, as described above, an individual at risk of developing a GPC3-related disorder, disease or condition would be a candidate for preventive therapy to prevent such disorder, disease or condition or one or more related clinical symptoms.

[0134] The prophylactic administration of immunotherapy, e.g., the prophylactic administration of an antibody disclosed herein or a composition comprising the antibody or a fragment thereof, can in certain embodiments provide protection to a recipient against a GPC3-related disorder, disease or condition, or reduce the risk of the recipient developing such disorder, disease or condition. In some alternative embodiments, prophylactic administration can reduce the likelihood of an increase in the severity of a GPC3-related disorder, disease or condition in a recipient.

[0135] The therapeutic administration of immunotherapy, e.g., the therapeutic administration of an antibody disclosed herein or a composition comprising the antibody or a fragment thereof, can inhibit the progression of a disorder, disease or condition and / or the clinical symptoms associated with the disorder, disease or condition. Such methods can include some embodiments that include reducing or inhibiting the expression of GPC3, the activity of GPC3, and / or the total amount of cells associated with GPC3 expression, which preferably results in the elimination of the disorder, disease or condition and its associated clinical symptoms.

[0136] In certain aspects, the present disclosure provides methods of treatment comprising co-administering or combining a GPC3 antibody or an antigen-binding fragment thereof with other cancer therapies. In a specific embodiment, the method comprises using a GPC3 antibody or an antigen-binding fragment thereof in combination with a therapy for treating liver cancer at different stages. Such liver cancer therapies are generally known in the art and include, but are not limited to, surgery (e.g., hepatectomy, liver transplantation), radiofrequency ablation (i.e., microwave / thermal ablation), percutaneous ethanol injection, radiotherapy (stereotactic body radiotherapy), chemoembolization, radioembolization, and / or targeted / systemic therapies. Non-limiting examples of targeted or systemic therapies include bevacizumab, atezolizumab, sorafenib, lenvatinib, cabozantinib, regorafenib, ramucirumab, pembrolizumab, nivolumab, ipilimumab, and various known combinations thereof.

[0137] The terms "subject", "individual", or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. For example, in the context of the present disclosure, mammals include 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 animals in a zoo). The term "animal" as used herein also includes humans. The term "subject" can also include a patient, i.e., an animal, and in certain embodiments, includes a human suffering from a disease associated with GPC3. A subject can also include a patient who may be at risk of developing a condition, disease, or disorder associated with GPC3. In certain embodiments, the subject, individual, or patient is a human.

[0138] Methods comprising an antibody or a binding fragment thereof described herein and a composition comprising the same can be administered by any conventional route, including injection or infusion, oral, buccal, sublingual, transdermal, intraocular, intranasal, aerosol, implant or depot, or rectal. In some embodiments, administration can be effected, for example, by injection (such as intravenous, intraperitoneal, intramuscular, subcutaneous, or transdermal).

[0139] The antibody, its fragment, and the composition comprising them are administered in an effective amount. "Effective amount" includes an amount that achieves the desired response or desired effect and can be in the form of a single dose or multiple doses. In the case of treating a particular disease or a particular clinical condition, the desired response involves inhibiting the progression of the disease or symptoms. Such inhibition can include slowing the progression of the disease / clinical symptoms and, in some embodiments, interrupting or reversing the progression of the disease / clinical symptoms. Treatment of a disease or disorder can also be delaying or preventing the onset of the disease or the disorder in a subject suffering from an active disease (such as cancer).

[0140] The effective amount of the composition of the present invention depends on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Thus, the dosage of the composition of the present invention can depend on various combinations of these parameters. In embodiments where the initial amount administered to the patient is insufficient, further administration can be effected at a higher amount, more frequent doses, or a different / more local route of administration.

[0141] In accordance with the above aspects, in some embodiments, the present disclosure relates to methods of monitoring a condition, disease, or disorder associated with GPC3 expression in a subject, wherein the methods comprise detecting and / or assaying or monitoring the amount of (i) GPC3 nucleic acid, (ii) GPC3 antigen or a portion thereof, (iii) an antibody against GPC3 or a portion thereof, and / or (iv) cells comprising GPC3 in a biological sample isolated from a patient. In some further embodiments, the condition, disease, or disorder associated with GPC3 expression comprises cancer.

[0142] In accordance with the above aspects, in some embodiments, the present disclosure relates to a method of diagnosing a condition, disease, or disorder characterized by GPC3 expression. In some embodiments, the method comprises detecting and / or assaying the amount of (i) GPC3 nucleic acid, (ii) GPC3 antigen or a portion thereof, (iii) an antibody against GPC3 or a portion thereof, and / or (iv) cells comprising GPC3 in a biological sample isolated from a patient. In some further embodiments, the condition, disease, or disorder associated with GPC3 expression comprises cancer.

[0143] In some embodiments of the above aspects, detecting comprises: (i) contacting a biological sample with an antibody or an antigen-binding fragment thereof disclosed herein, wherein the antibody or the antigen-binding fragment thereof specifically binds to GPC3 or a portion thereof, an anti-GPC3 antibody or a portion thereof, and / or at least a portion of a cell surface-expressing GPC3 antigen; and (ii) detecting the formation of a complex between the antibody or the antigen-binding fragment thereof and GPC3 or a portion thereof, an anti-GPC3 antibody or a portion thereof, and / or a cell comprising the GPC3 antigen. In some embodiments, a biological sample isolated from a patient is compared with a reference sample (e.g., a comparable biological sample obtained from a healthy subject having a normal GPC3 expression level).

[0144] In another aspect, the present disclosure relates to a method for determining the regression, course, or onset of a disease characterized by GPC3 expression. In some embodiments, the disease can be identified according to the various methods provided by the present disclosure. In some embodiments, the method comprises monitoring one or more parameters in a patient sample from a patient having or suspected of having the disease, the parameters selected from: (i) the amount of nucleic acid associated with GPC3; (ii) the amount of expressed GPC3 antigen or a portion thereof; (iii) the amount of an antibody against the GPC3 antigen or a portion thereof; and / or (iv) the amount of cells associated with GPC3 expression. In some embodiments, the method comprises assaying the (one or more) parameters in a first sample at a first time point and in another sample at a second time point, wherein the course of the disease is determined by comparing the two samples.

[0145] The term "sample" can include any sample useful or available according to the methods described herein. In some embodiments, the sample can be a biological sample, such as a sample from tissue, which can include, for example, body fluids and / or cell samples (e.g., tumor / tissue biopsies, blood draws, bronchial aspirates, sputum, urine, feces, or other body fluids) that can be obtained by any conventional method. In some embodiments, the term "sample" can include processed samples, such as fractions or isolates of biological samples, such as nucleic acid and peptide / protein isolates.

[0146] According to some embodiments, one or more antibodies, constructs, or fragments described herein can be used to detect the GPC3 antigen or related cells expressing the GPC3 antigen or fragments thereof, or to determine or monitor the amount of the GPC3 antigen or cells related to at least a portion of the GPC3 antigen. Methods that include detection using the antibodies or fragments thereof disclosed herein can include a detectable label. In some embodiments, the detectable label or tag is a fluorescent label, a colorimetric label, a radioactive label, or an enzyme label, or other labels disclosed herein or known in the art.

[0147] As described above, the methods provided herein can reduce, slow, or stop tumor growth, and in certain aspects, such reduction or slowing can be statistically significant. The reduction in tumor growth can be measured by comparison to the patient's baseline tumor growth, expected tumor growth, expected tumor growth based on a large patient population, or tumor growth in a control population.

[0148] In certain aspects, administration of a GPC3 antibody or an antigen-binding fragment thereof can increase overall survival (OS). In other aspects, administration of a GPC3 antibody or an antigen-binding fragment thereof can result in stable disease (SD).

[0149] F. Kits

[0150] Another aspect of the present disclosure is a kit. In one aspect, the kit contains any of the sequences, compounds, and pharmaceutical formulations or compositions of nucleic acids, polypeptides, expression vectors, or host cells generally described above, as well as instructions or labels for guiding proper use or administration. Optionally, the kit may also contain one or more containers, reagents, reactants, and / or syringes or other devices to facilitate delivery or use. The present disclosure contemplates that all components or any subset of components for performing research assays, diagnostic assays, and / or for administering a therapeutically effective amount may be placed in the kit. Similarly, the kit may contain instructions for preparing an amino acid sequence / polypeptide, for example, by culturing a host cell expressing a nucleic acid encoding an antibody (or a construct or an antigen-binding fragment thereof) of the present disclosure under suitable conditions to prepare the amino acid sequence / polypeptide. For another example, a kit for therapeutic administration of an antibody of the present disclosure may contain a solution containing the antibody pharmaceutical formulation or a lyophilized product of the antibody, as well as instructions for administering the composition to a patient in need and / or for reconstituting the lyophilized product.

[0151] The present disclosure also encompasses finished packaged and labeled pharmaceuticals. The article includes a suitable unit dosage form placed in a suitable vessel or container (such as a glass vial or other sealed container). For dosage forms suitable for parenteral administration, the active ingredient (such as the above-mentioned antibody) is sterile and suitable for administration in a particle-free solution form. In certain aspects, the formulation is suitable for intravenous administration, for example, for intravenous infusion to a human or an animal.

[0152] In a specific aspect, the formulation of the present disclosure is formulated in a single-dose vial in a sterile liquid form. Exemplary containers include, but are not limited to, vials, ampoules, pre-filled syringes, IV bags, and blister packs (containing one or more pills). Such containers may optionally be attached with a notice in a form prescribed by a government agency responsible for regulating the production, use, or sale of pharmaceuticals or biological products, and the notice reflects obtaining approval from the said agency regarding the production, use, or sale for human diagnosis and / or administration.

[0153] Like any pharmaceutical, the packaging materials and containers are designed to protect the stability of the product during storage and transportation. In addition, the products of the present disclosure include instructions for use or other information materials for advising doctors, technicians, or patients on how to appropriately prevent or treat related diseases or disorders. In other words, the article includes instructions or suggestions for a dosing regimen, including but not limited to the actual dose, monitoring procedures, etc., as well as illustrative tools for other monitoring information.

[0154] A kit for a diagnostic assay may comprise a solution containing an antibody or an antigen-binding fragment thereof of the present disclosure or a lyophilized product of an antibody or a fragment thereof of the present disclosure (wherein the antibody or fragment specifically binds to one or more targets), and a reagent for detecting such an antibody. The antibody may be labeled according to methods known in the art and described herein, including but not limited to labels such as small molecule fluorescent tags, proteins such as biotin, GFP or other fluorescent proteins, or epitope sequences such as his or myc. Similarly, a first antibody for detecting the antibody may be included in the kit. The first antibody may be directed against a sequence on the antibody, or against a label, tag or epitope used to label the antibody. The first antibody may in turn be labeled for detection, or, if further signal amplification is desired, the first antibody may be detected by a second antibody, which may also be included in the kit.

[0155] Kits for research use are also contemplated. Such kits may be, for example, similar to kits for diagnostic or therapeutic use, but further include a label specifying that the kit and its use are limited to research purposes.

[0156] G. Modification and / or Engineering

[0157] Another aspect of the present disclosure includes modifying and / or engineering the antibodies and / or their antigen-binding fragments described herein. For example, antibodies and / or their antigen-binding fragments include but are not limited to chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies, diabodies, multispecific antibodies, dual-specific antibodies, and bispecific antibodies. Non-limiting embodiments of such constructs and antibody forms are illustrated in the Examples and throughout the present disclosure. The antibodies and antigen-binding fragments thereof of the present disclosure may also be used to generate fusion proteins and / or for cell fusion targeted by the antibody, such as fusion with an immunological fusion partner and a cell.

[0158] The disclosed antibodies and their antigen-binding fragments may also be used to prepare therapeutic immunoconjugates, wherein the disclosed antibody or its antigen-binding fragment is conjugated to one or more therapeutic agents. Non-limiting embodiments of such immunoconjugates are illustrated throughout the present disclosure. In addition, for example, the disclosed antibody or its antigen-binding fragment may be used to prepare antibody-drug conjugates (ADCs). Drugs that may be used include but are not limited to cell proliferation inhibitors, anti-cancer agents, drugs that induce apoptosis, and combinations thereof.

[0159] As described herein, unless otherwise indicated, the practice of the methods disclosed herein employs available techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are familiar to those skilled in the art. These techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991).

[0160] The following examples illustrate some of the above aspects and embodiments, but are not intended to limit the scope of the claimed invention.

[0161] Examples

[0162] Antibody Generation, Phage Display, and Affinity Maturation

[0163] Antibody generation was performed using glypican 3 (GPC3) as the panning antigen (Sino Biological, Cat#10088-H08H-B). A library was constructed containing IgM (1×10 5 -2×10 9 colonies per round) or IgG (1×10 5 -2×10 8A fully human scFv library (from [number of colonies]) was panned against the GPC3 antigen. After three rounds of panning, the clones positive in phage ELISA were sequenced. More than 40 unique sequences were obtained from over 200 sequenced clones. These sequences were converted to scFv-Fc for expression and functional screening. The following examples provide details of 8 representative clones that have been deeply characterized. One of the 8 representative clone sequences was used to develop a series of affinity-matured constructs (using phage display), which focused on modification of the VH region to screen and evaluate improved GPC3 binding affinity.

[0164] Nine affinity-matured clones were selected for further characterization. The resulting data detailed below demonstrate that the present disclosure provides antibodies (i.e., GPC3-binding antibody constructs / fragments) with excellent GPC3-specific binding properties and provides their use alone or as immunoconjugates for various applications, such as for therapeutic methods (e.g., methods for inhibiting / neutralizing GPC3 biological activity, methods for treating cancer) and for GPC3 assay and diagnostic applications.

[0165] Example 1 - Generation and Screening of IgG Binding to GPC3 Ligand

[0166] Direct ELISA assay is the main method used to define the binding specificity of mAbs identified from a fully human scFv phage library.

[0167] From the scFv library, 16 candidates were screened for expression in mammalian expression host cells in the form of scFv-Fc and isolated and purified for functional analysis. The heavy chain variable region (VH) and light chain variable region (VL) sequences were fused to the human IgG1 constant region and transiently transfected into 293F cells.

[0168] For ELISA analysis, 96-well ELISA plates (Thermo, Cat#3855, Immulon 4HBX) were coated overnight at 4°C with recombinant human glypican-3 protein (His-tag) (SinoBiological, Cat#10088-H08H) at 1 μg / ml in phosphate-buffered saline (PBS). The plates were then incubated with supernatants containing scFv-Fc expressed by HEK293 cells, where the supernatants were serially diluted in PBST buffer (PBS + 0.1% Tween) + 3% milk powder. A secondary antibody, goat anti-human Fc-HRP (Invitrogen, Cat#A18829), was added at a final concentration of 0.5 μg / ml in PBST + 3% milk powder. All steps were incubated for 1 hour at room temperature. Between each incubation step, the plates were washed three times with PBST. After incubation with the secondary antibody, HRP substrate 1-step Ultra TMB-ELISA (Thermo, Cat#34029) was added to generate a colorimetric signal. The HRP reaction was terminated by adding 2 M sulfuric acid. Measurements (OD 450 ) were performed using a SpectraMax M3 microplate reader (Molecular Devices, CA, USA). The ELISA binding results were ranked, and the best binders were selected for further analysis.

[0169] Table 1 details the sequence information of several exemplary GPC3-binding antibody constructs according to the embodiments disclosed herein.

[0170] Table 1. Heavy and light chain variable region sequences of representative GPC3 antibodies and the identified CDR regions (bold, underlined) Candidate

[0171]

[0172]

[0173] Example 2 - GPC3 Binding

[0174] Using Octet QKe (ForteBio), a three-step protocol was adopted to measure the binding affinity. In the first step, seven anti-hIgG Fc capture (AHC) biosensor probes were loaded by contacting them with seven different antibody solutions (2.5 μg / ml, in different microplate wells respectively) of the test antibody. The reference probes were loaded with an equal amount of control antibody. In the second step, the loaded biosensor probes were contacted with the antigen in wells containing a series of antigen concentrations (0 nM, 5.625 nM, 11.25 nM, 22.5 nM, 45 nM, 90 nM, 180 nM, and 360 nM). Another separate antigen well (360 nM) was set up for the reference probes. The reaction was monitored for 300 seconds. In the third step, the probes were transferred to a buffer solution to initiate the dissociation reaction. The dissociation reaction was monitored for 600 seconds. After measurement, the probes were regenerated according to the manufacturer's instructions. The obtained data was fitted to a 1:1 binding model using Octet data analysis software (see Table 2).

[0175] Table 2. K of the selected candidates measured by Octet D value

[0176] Ligand Hu-GPC3(10088-H08H) <![CDATA[K D > P1 4.76 nM Cy-GPC3(GP3-C5225) P1 1.84 nM Hu-GPC3(10088-H08H) P3 20.1 nM Cy-GPC3(GP3-C5225) P3 5.45 nM Hu-GPC3(10088-H08H) P10 2.85 nM Cy-GPC3(GP3-C5225) P10 1.28 nM Hu-GPC3(10088-H08H) P17 9.1 nM Cy-GPC3(GP3-C5225) P17 6.01 nM Hu-GPC3(10088-H08H) P20 100 nM Hu-GPC3(10088-H08H) 59 11.3 nM Cy-GPC3(GP3-C5225) 59 6.49 nM Table 3. GPC3

[0177] Example 3 - Epitope binning

[0178] Using sandwich epitope binning, it was detected whether the exemplary antibodies could block each other's binding to the GPC3 antigen epitope. Briefly, the first anti-GPC3 scFv-Fc was diluted to 10 nM in buffer and captured by the AHC biosensor. Then the biosensor was transferred to an antigen well containing 300 nM human glypican 3 protein (SinoBiological, Cat#10088-H08H) in the same buffer to initiate antigen binding (Phase I binding). After this initial binding, the biosensor was immersed in another well containing 200 nM of the second anti-GPC3 scFv-Fc to initiate Phase II binding. Compared with Phase I, the binding rate in Phase II was slower, indicating epitope competition (e.g., blocking binding). Antibodies that blocked binding to the same epitope were grouped together. The results showed that P17, P19, and P20 had substantially similar epitopes, P1 and P10 had common but different epitopes, while P3, P59, and P71 each seemed to bind a unique epitope (Table 3).

[0179] Grouped data of scFv-Fc Group

[0180] Construct P17, P19, P20 1 P1, P10 2 P3 3 Figure 1 4 59 5 71

[0181] Example 4 - Cell Surface Binding

[0182] Glypican-3 protein is expressed on the surface of mammalian cells, so binding values can be evaluated and analyzed by cell-based assays. GPC3-positive HepG2 cells and GPC3-negative NCI-H1975 and NCI-H929 cells were used for cell surface binding assays. Cells were seeded at 1×10 5 to 5×10 5 cells per well in 96-well plates and incubated with anti-GPC3 scFv Fc fusion protein on ice for 45 minutes. Cells were washed twice and then (at 1:1000) goat anti-human IgG (H+L) conjugated to AlexaFluor TM 647 (Invitrogen, Cat# A21445) was added to detect the bound scFv-Fc. Cell surface-bound fluorescence was measured and analyzed using Attune NxT flow cytometry (Thermo Fisher Scientific). Migration of the fluorescence signal in GPC3-positive HepG2 cells confirmed cell surface binding of candidates P1, P3, P10, P17, P19, P20, 59, and 71, while no migration of the fluorescence signal was observed in GPC3-negative NCI-H1975 and NCI-H929 cells, indicating ligand specificity of these candidates (see Figure 3A - Figure 3B ).

[0183] Example 5 - Cell Internalization

[0184] Ligand internalization via receptor-mediated endocytosis can be used to determine whether an extracellular molecule binds to its specific receptor protein on the cell surface. To evaluate ligand internalization of exemplary GPC-antibody constructs in cells expressing GPC3, the scFv was converted to the Fab form and incubated with HepG2 and Huh7 cells. The Fab was diluted to 100 μg / mL with cold FACS buffer. In 96-well plates, P3, P10, P17, P20, 59, and 71 Fab (100 μL) or control (100 μL FACS buffer) were added to separate wells containing HepG2 and Huh7 cells, respectively, and incubated on ice for 30 minutes to allow the antibody (Fab) to bind to all antigen sites on the target cells.

[0185] Except for the control sample (t = 0 h), cells were incubated at 37 °C to induce endocytosis of the binding protein. Samples were harvested at different time points (0 h, 1 h, 2 h, 3 h, and 4 h) and immediately washed twice with cold FACS buffer to remove any unbound Fab. Samples were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After collecting all samples, 1 μl of FITC-fluorescently labeled anti-human IgG (H+L) secondary antibody was added to each sample and incubated at 4 °C for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 100 μl of FACS buffer for analysis using a CytoFLEX flow cytometer (Beckman Coulter). The presence of internalized Fab (int.Fab) was represented by a decrease in median fluorescence intensity (MFI) using the following formula:

[0186] MFI of Int.Fab = MFI of 0-hour sample - MFI of sample at different time points.

[0187] As shown in Figure 2, the amount of internalized Fab increased over time, and different internalization rates were observed between different cells (HepG2 (upper panel) and Huh7 (lower panel)). The higher MFI observed in HepG2 indicates a higher amount of GPC3 ligand expressed on the surface of HepG2 cells compared to Huh7 cells. The increase in MFI over time indicates more internalization in HepG2 and Huh7 cells. Although various factors may contribute to this observation, the higher average internalization rate typically associated with HepG2 cells compared to Huh7 cells may be related to the higher expression level of GPC3 in HepG2 cells.

[0188] Example 6 – Cancer cell inhibition

[0189] An immunoconjugate of a GPC3-binding molecule was recombinantly prepared as a candidate immunotoxin (RIT) molecule, and the immunoconjugate was constructed as a Fab conjugated to a Pseudomonas exotoxin A fragment. This RIT binds to GPC3 on the cell surface via the Fab fragment and inhibits protein synthesis in target cells after internalization, thereby inducing apoptosis. The cell proliferation inhibitory effect and cytotoxicity of the P10, P17, 59, 71 RIT constructs were evaluated on HepG2 and Huh7 cell lines by CCK8 assay.

[0190] Briefly, HepG2 and Huh7 cells were seeded in 96-well plates at a density of 5000 cells per well and cultured for 24 hours. After attachment, different concentrations of P10, P17, 59, 71RIT (25, 1250, 5000, 15000, 25000, 40000, 50000, 100000, 200000, 400000 ng / ml) were added to the cells in triplicate and incubated for 72 h under standard culture conditions. After 72 h, the medium was aspirated, and 10 μL of CCK8 solution and 90 μL of fresh medium were added to each well. The plate was incubated at 37 °C for 0.5 - 1 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Tecan), and the percentage of cell survival was calculated according to the following formula: (live cells)% = (OD value of immunotoxin - OD value of blank sample / OD value of control - OD value of blank sample) × 100. Nonlinear regression analysis was performed using the fitting formula of log(inhibitor) vs. response (three parameters) (GraphPad Prism 8.0.2, San Diego, California) to analyze the IC 50 value.

[0191] As Figure 3A shown, GPC3 RITs showed dose-dependent inhibitory effects on the proliferation of HepG2 ( Figure 3B ) and Huh7 ( P17 RIT ) cell lines. Table 4 summarizes the two RITs with the lowest measured IC 50 values. The data indicate that RITs constructed using GPC3-binding sequences can be successfully internalized and used as therapeutic agents for diseases associated with uncontrolled cell proliferation, such as cancer. Different cytotoxicity results were observed for different RITs constructed using different lead candidate sequences with different binding affinities.

[0192] Table 4. Inhibitory effects of immunoconjugates

[0193] <![CDATA[IC50 against HepG2 50 > <![CDATA[IC for Huh7 50 > 59.9 nM 231.3 nM 59RIT 6.5 nM 99.36 nM Figure 4

[0194] Example 7 - Apoptosis induction by immunoconjugates

[0195] The ability of GPC3 RIT to induce apoptosis / necrosis in HepG2 cells was analyzed using Annexin V and PI staining. Briefly, HepG2 cells were seeded in 12-well plates overnight (2 × 10 5cells / well). After adhesion, RITs (P3, P10, P17, 59, 71) were diluted to concentrations of 5000, 10000, 15000, 30000 ng / ml with cell culture medium and added to the wells, followed by incubation for 24 hours. After incubation, the cells were digested and collected with trypsin (without EDTA), washed twice with pre-chilled PBS, and then resuspended in pre-chilled 1× binding buffer (Annexin V-FITC / PI apoptosis kit, Multi sciences). The blank / control group was divided into two tubes. The first tube was incubated with 500 μL of apoptosis positive control solution, and the second tube was incubated on ice in pre-chilled PBS for 30 min. After washing twice with pre-chilled PBS, 1 μL of Annexin V-FITC or 2 μL of PI was added to the control group and the experimental group respectively, gently mixed, and incubated in the dark at room temperature for 5 min. The samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The proportion of apoptotic cells was calculated as follows: % apoptosis rate = (% Annexin-V positive cells + % double positive cells) / total number of cells × 100.

[0196] As 74 pM shown, GPC3 RITs induced apoptosis in HepG2 cells in a dose-dependent manner, indicating that the RITs constructed with GPC3-binding sequences were successfully internalized and verified the mechanism of action that could be used for cancer treatment. Different RITs constructed with different lead sequences with different binding affinities resulted in different cytotoxicities.

[0197] Example 8 - T Cell Activation of GPC3-CD3 Bispecific Antibody Constructs

[0198] A series of bispecific antibodies were constructed using the GPC3 antibody sequences and CD3 antibody scFv described herein. The effects of these bispecific antibodies on bridging cells expressing GPC3 and activating CD3-positive T cells were evaluated. Briefly, through engineering, the CD3 scFv was placed at the C-terminus of the GPC3 mAb light chain and linked by a linker sequence. The bispecific antibody was expressed in 293F cells and recovered and purified using a MabSelect SuRe column (Cytiva, Cat#28926977). Target cells expressing GPC3 (e.g., HepG2, 25,000 cells / well) and effector cells (e.g., Jurkat, 120,000 cells / well) were seeded in 96-well plates. The purified bispecific antibody was added to the cell cultures at different concentrations, incubated for 7 hours, and then the end-point readings of T cell activation were recorded.

[0199] Wells inoculated with effector cells but not with GPC3 target cells served as negative controls. Bispecific antibodies constructed from the variable domains of P1, P3, P10, P17, and 59, in combination with CD3 scFv, successfully induced strong activation of T cells in the presence of GPC3-positive target cells, while no T cell activation or very low T cell activation was induced in the presence of GPC3-negative NCI-H1975 cells or in the absence of target cells, as summarized in Table 5.

[0200] Table 5. T cell activation efficacy of GPC3-CD3 bispecific antibodies

[0201] P1 P3 P10 P17 59 <![CDATA[EC for CHOK1 / GPC3(++) 50 > 171 pM 91 pM 63 pM 63 pM 137 pM <![CDATA[EC for HepG2(+) 50 > 182 pM 85 pM 40 pM 68 pM 102 pM <![CDATA[EC for Hep3B(+) 50 > 314 pM 40 pM 27 pM 49 pM >20 nM <![CDATA[EC for NCI-H1975(-)]]> 50 > >64 nM >12 nM >50 nM >79 nM Figure 5

[0202] Example 9 – Affinity maturation

[0203] Using degenerate oligonucleotides, random mutations were introduced into the CDRs of the above heavy and light chain variable region sequences (see Table 1), resulting in a total of 11 libraries. The design of the oligonucleotides is summarized in Table 6. The libraries constructed to cover the same CDRs were pooled for panning. Except for the library covering LCDR3, each library covering other CDRs was subjected to two rounds of panning. Briefly, biotinylated GPC3 (Sino Biological, Cat#10088-H08H-B) was added to M-280 streptavidin Dynabeads TM (Invitrogen, Cat#11205D), and then incubated with the phage library. Unbound and weakly bound phages were washed away with PBST (PBS containing 0.1% Tween-20). Phages displaying scFv with relatively high affinity remained on the beads, were eluted with 0.1 N HCl, and rescued with log-phase TG1 cells. Single colonies of these cells were randomly picked and sent for sequencing. After one round of panning, most of the sequenced colonies from the LCDR3 library had the same sequence as "WT", indicating that the LCDR3 sequence might play a key role (non-mutable) in the GPC3 antibody-antigen interaction. Therefore, the LCDR3 sequence was kept unchanged.

[0204] Table 6. Oligonucleotide design

[0205]

[0206]

[0207] To identify specific CDRs that contribute more to GPC3 binding and thus further improve binding, soluble scFv ELISA was performed on each library. Single colonies were picked and inoculated onto 96-well round bottom plates containing 2x YT + 100 μg / mL carbenicillin + 2% glucose and amplified overnight. After amplification, the overnight cultures were inoculated into fresh 2x YT + 100 μg / mL carbenicillin, induced with 1 mM IPTG, and treated with 5000 units / mL polymyxin B to release the scFv. The clarified supernatant containing the soluble scFv was obtained by centrifuging the cells.

[0208] The scFv ELISA was generally performed as follows. Immulon 4HBX plates (Thermo Scientific, cat#3855) were coated with 1 μg / mL human GPC3 (Sino Biological, cat#10088-H08H) in PBS overnight at 4°C. The plates were washed 3 times with PBST (PBS + 0.1% Tween-20), then blocked with 4% non-fat milk in PBST, and incubated with a 1:1 dilution of the supernatant containing the scFv for 1 hour (at room temperature). The bound scFv was detected with a 1:2000 dilution of the c-Myc antibody (Invitrogen, cat#MA1-980-1MG) and a 1:1000 dilution of the mouse IgG HRP-conjugated secondary antibody (R&D Systems, cat#HAF007). Each incubation step was carried out for 1 hour at room temperature. The plates were washed three times with PBST between each incubation step. Color development was carried out by adding 1-Step TM TMB-ELISA substrate (Thermo Scientific, cat#34029), and the reaction was then stopped with 2M sulfuric acid. Then, OD 450 values were obtained by SpectraMax M3 (Molecular Devices, CA, USA).

[0209] Positive clones were defined as clones with OD 450 values 2-fold higher than P17 WT and were sequenced. The positive rates of each CDR are listed in Table 7. The positive rate of the heavy chain CDR was higher than that of the light chain CDR, indicating that mutations on the heavy chain CDR may benefit more from affinity maturation than those on the light chain CDR. It can be seen that affinity maturation mainly focuses on the heavy chain CDR.

[0210] Table 7. CDR and positive rate

[0211]

[0212]

[0213] It is generally known that HCDR3 plays an important role in antigen recognition. The VH sequence of P17 was re-analyzed using the proABC-2 online program (website: bianca.science.uu.nl / proabc2 / ) on the Utrecht Biomolecular Interaction Web Portal. This web server program estimates the probability of interaction of each residue with the cognate antigen based on a deep learning framework. The prediction results are as Figure 6 shown. Based on these prediction results and the sequencing results of the positive clones from the initial HCDR3 library, two new HCDR3 libraries were constructed. Generally, two rounds of panning were carried out according to the procedure discussed above in this section. Clones identified by ELISA (high OD 450 reading) were sequenced.

[0214] Another library was constructed, which focused on the beneficial mutations identified by the previous sequencing results while reducing the size of the library. In addition, to reduce the potential immunogenicity of P17, mutagenesis was carried out on HCDR1 and HCDR2, with the overall goal of reverting these two CDRs back to the germline sequence and improving antibody affinity. All degenerate oligonucleotides are listed in Table 8. To improve the binding affinity, the three heavy-chain CDR libraries were combined in the following order: (1) The HCDR1 and HCDR2 libraries were combined and subjected to two rounds of panning against GPC3; (2) The HCDR3 focused library after one round of panning was combined with the panned HCDR1-HCDR2 combined library; (3) The heavy-chain shuffled library was subjected to two more rounds of panning against GPC3. After that, more than 600 single colonies were picked from this HCDR3 focused library and this heavy-chain shuffled library before and after the second round of panning. Using the same procedure as above, soluble scFv was prepared and analyzed by ELISA. Among the 56 sequenced clones, 9 clones with unique sequences and the highest OD 450 reading in ELISA (P17-8E12, P17-11F4, P17-13F3, P17-13G2, P17-15D5, P17-15F8, P17-16A1, P17-16A11, P17-16E6) were selected as high-affinity lead molecules.

[0215] Table 8. Oligonucleotides

[0216]

[0217]

[0218] Example 10 - GPC3 Binding of Affinity-Matured Constructs

[0219] Using the three-step protocol described above (i.e., using Octet QKe (ForteBio, Torrance, CA)), the antibody binding affinities of the affinity matured sequences were measured. Step 1: Prepare anti-human Fc capture (AHC) biosensor probes by loading the test antibody (2.5 μg / mL), and prepare reference biosensor probes using an equal amount of control antibody. Step 2: Contact the probes with serial dilutions of the antigen (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0 nM GPC3, SinoBiological) for 600 seconds. Another 100 nM antigen well was set up for the reference probe. Then transfer the probes to the kinetic buffer (see above) to allow dissociation for 300 seconds. Fit the data to a 1:1 binding model using Octet data analysis software.

[0220] The binding kinetics of P17 WT and five affinity matured constructs are as Loaded sample shown, and the calculated K D values are shown in Table 9. The affinity matured constructs showed a significantly improved K D value (i.e., order of magnitude), which may be associated with a significantly slower dissociation rate. For example, the K D values of P17-8E12 and P17-11F4 reached the sub-nanomolar level and were calculated to be 6000-fold lower than the K D value of P17 WT.

[0221] Table 9. Binding of affinity matured constructs

[0222] Ka (1 / Ms) <![CDATA[K D (M)]]> Kdis (1 / s) P17 WT IgG P17-8E12IgG 5.99E-09 1.39E+05 8.33E-04 P17-11F4 IgG <1.0E-12 2.23E+05 1.79E-07 P17-16A1 IgG <1.0E-12 1.88E+05 1.83E-07 P17-16A11IgG 1.17E-12 2.15E+05 2.51E-07 P17-16E6 IgG 1.03E-12 1.82E+05 1.87E-07 Figure 7 1.33E-12 2.08E+05 2.76E-07

[0223] All affinity matured constructs were compared with P17 WT using ELISA. Briefly, Immulon4HBX plates (Thermo Scientific, Cat#3855) were coated with 1 μg / mL Fc-tagged human glypican 3 protein (Acro Biosystems, Cat#GP3-H5258-100ug) in PBS overnight at 4 °C. After washing three times with PBST (PBS + 0.1% Tween-20), the plates were blocked with 4% non-fat milk (in PBST) and incubated with serial dilutions of the Fab constructs. Bound Fab was detected using a 1:1000 dilution of HRP-conjugated anti-λ light chain antibody (Abcam, Cat#ab9007). Incubation for each step was 1 hour at room temperature. Between each incubation step, the plates were washed three times with PBST. By adding 1-Step TMThe TMB-ELISA substrate (Thermo Scientific, Cat# 34029) was developed, and then the reaction was terminated with 2 M sulfuric acid. OD 450 values were collected by SpectraMax M3 (Molecular Devices, CA, USA).

[0224] Figure 8 ELISA binding curves of titrated P17 WT and the 9 affinity matured constructs analyzed are shown. Compared to P17 WT, the EC 50 values of all constructs were significantly lower (ranging from 8.4 - 11.7 ng / mL), and the maximum response values were higher (Table 10). These results indicate that the binding ability of the affinity matured constructs to recombinant human GPC3 was improved.

[0225] Table 10. EC50 of affinity matured constructs

[0226]

[0227] Example 11 - Cell surface binding of P17 affinity matured constructs

[0228] Flow cytometry was used to screen the affinity matured P17 constructs to determine their cell surface binding activity to endogenously expressed GPC3. Briefly, Hep3B cells (ATCC, Cat# HB-8064) were removed from the culture flask and blocked with PBS + 1% FBS on ice for 1 hour. The cells were diluted (2 x 10 5 cells / well) in a 96-well plate and stained with serial dilutions of P17 WT and the affinity matured constructs (Fab form) on ice for 1 hour. After washing twice with PBS + 1% FBS, the Fab bound to the cell surface was detected with Alexa-647 labeled anti-human IgG (H+L) antibody (Invitrogen, cat# A21445, 1:1000 dilution). The cells were washed twice and then stained on ice with 1:1000 diluted SYTOX Green dead cell dye (Invitrogen, Cat# S34860) to distinguish dead cells from live cells. After removing the free dye by two rounds of washing, the cells were analyzed on a flow cytometer.

[0229] Figure 9 The cell surface binding profiles shown indicate that the binding of the affinity matured P17 constructs to cell surface expressed GPC3 was improved relative to P17 WT, with the EC50 values of most constructs being below 10 ng / mL (see Table 10 above).

[0230] The breast cancer cell line that is known not to express GPC3 (T-47D, ATCC, Cat# HTB-133) was used to evaluate whether any non-specific binding ability was generated by the affinity maturation construct during the maturation process. The T-47D cells were removed from the culture flask and blocked on ice for 1 hour with PBS + 1% FBS. The cells were seeded at 2x10 5 cells / well in a 96-well plate and stained with the P17 wild type and the affinity maturation construct (in IgG form) at a concentration of 9 μg / mL. Rituximab, an FDA-approved drug known to specifically bind to CD22, was used as a negative control for the experiment. Bound IgG was detected using a 1:1000 dilution of a PE-conjugated goat anti-human IgG Fc secondary antibody (Invitrogen, Cat# 12-4998-82). Dead cells were stained with a 1:1000 dilution of LIVE / DEAD TM Fixable Far Red (Invitrogen, Cat# L34973). All incubation steps were carried out on ice for 1 hour. Between each incubation step, the cells were washed twice with PBS + 1% FBS to remove any free antibody or dye. The fluorescence signal was detected on a flow cytometer.

[0231] The histograms of all nine candidate drugs overlapped with the histogram of the negative control, indicating that none of the candidate drugs had non-specific binding activity to GPC3-negative T-47D cells.( ​ )

[0232] Table 11: Amino acid sequences of the heavy chain variable regions and CDRs of representative affinity maturation GPC3 constructs.

[0233]

[0234]

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to glypican-3 (GPC3) protein, said antibody or antigen-binding fragment thereof comprising: i. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:1; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:3; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4; iii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:6; iv. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:7; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; v. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:9; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:10; vi. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:11; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:12; vii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:13; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:14; viii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:15; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:48; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; x. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:52; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:56; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:60; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:64; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:68; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:72; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xvi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:76; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; or xvii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:80; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising: i. A heavy chain variable region comprising SEQ ID NO:1; and A light chain variable region comprising SEQ ID NO:2; ii. A heavy chain variable region comprising SEQ ID NO:3; and A light chain variable region comprising SEQ ID NO:4; iii. A heavy chain variable region comprising SEQ ID NO:5; and A light chain variable region comprising SEQ ID NO:6; iv. A heavy chain variable region comprising SEQ ID NO:7; and A light chain variable region comprising SEQ ID NO:8; v. comprising a heavy chain variable region of SEQ ID NO:9; and a light chain variable region of SEQ ID NO:10; vi. comprising a heavy chain variable region of SEQ ID NO:11; and a light chain variable region of SEQ ID NO:12; vii. comprising a heavy chain variable region of SEQ ID NO:13; and a light chain variable region of SEQ ID NO:14; viii. comprising a heavy chain variable region of SEQ ID NO:15; and a light chain variable region of SEQ ID NO:16; ix. comprising a heavy chain variable region of SEQ ID NO:48; and a light chain variable region of SEQ ID NO:8; x. comprising a heavy chain variable region of SEQ ID NO:52; and a light chain variable region of SEQ ID NO:8; xi. comprising a heavy chain variable region of SEQ ID NO:56; and a light chain variable region of SEQ ID NO:8; xii. comprising a heavy chain variable region of SEQ ID NO:60; and a light chain variable region of SEQ ID NO:8; xiii. comprising a heavy chain variable region of SEQ ID NO:64; and a light chain variable region of SEQ ID NO:8; xiv. comprising a heavy chain variable region of SEQ ID NO:68; and a light chain variable region of SEQ ID NO:8; xv. comprising a heavy chain variable region of SEQ ID NO:72; and a light chain variable region of SEQ ID NO:8; xvi. comprising a heavy chain variable region of SEQ ID NO:76; and a light chain variable region of SEQ ID NO:8; or xvii. comprising a heavy chain variable region of SEQ ID NO:80; and a light chain variable region of SEQ ID NO:

8.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment comprises a single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), single-chain antibody, single-domain antibody, Fab fragment or F(ab')2 fragment.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody comprises an IgG, IgM, IgA, IgE, IgD or IgY isotype.

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1, 2 or 4, wherein the antibody is a monoclonal antibody (mAb).

6. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-5, which further comprises a conjugate moiety.

7. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the conjugate moiety comprises a therapeutic agent, solid support, affinity agent or detectable label.

8. The isolated antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the conjugate moiety comprises an anti-cancer agent or cytotoxin.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8, which binds to glypican-3 protein with a K D value of about 1.0 pM to 200 nM.

10. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8 inhibits cell proliferation with an IC50 value of about 0.01-250 nM.

11. A bispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, and an antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain the GPC3 epitope.

12. The bispecific antibody according to claim 11, wherein the antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain the GPC3 epitope comprises a therapeutic antibody.

13. The bispecific antibody according to claim 11, wherein the antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain the GPC3 epitope induces an immune response.

14. The bispecific antibody according to claim 11, wherein the antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain the GPC3 epitope binds to an antigen comprising CD2, CD3, CD11a, CD20, CD25 (IL2R), CD28, CD33, CD47, CD52, EGFR, VEGF, integrin-α3, GPIIb / IIIar, Protein F, TNF-α, TNF-β, HER2 / Neu, C5 or IgE.

15. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-5, which is conjugated to a solid support, an affinity agent or a detectable agent.

16. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. A kit comprising the pharmaceutical composition according to claim 16, or the antibody or antigen-binding fragment thereof according to any one of claims 1-15, optional reagents and instructions for use.

18. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to glypican-3 (GPC3) protein, the antibody or antigen-binding fragment thereof comprising: i. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:1; and A light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:3; and A light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:4; iii. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:5; and A light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:6; iv. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:7; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; v. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:9; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:10; vi. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:11; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:12; vii. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:13; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:14; viii. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:15; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:48; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; x. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:52; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:56; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:60; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:64; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 68; and a light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 8; xv. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 72; and a light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 8; xvi. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 76; and a light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 8; or xvii. A heavy chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 80; and a light chain variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

19. The method according to claim 18, wherein the antibody or antigen-binding fragment thereof of claim 1 comprises: i. A heavy chain variable region comprising SEQ ID NO: 1; and a light chain variable region comprising SEQ ID NO: 2; ii. A heavy chain variable region comprising SEQ ID NO: 3; and a light chain variable region comprising SEQ ID NO: 4; iii. A heavy chain variable region comprising SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 6; iv. A heavy chain variable region comprising SEQ ID NO: 7; and a light chain variable region comprising SEQ ID NO: 8; v. A heavy chain variable region comprising SEQ ID NO: 9; and a light chain variable region comprising SEQ ID NO: 10; vi. A heavy chain variable region comprising SEQ ID NO: 11; and a light chain variable region comprising SEQ ID NO: 12; vii. A heavy chain variable region comprising SEQ ID NO: 13; and a light chain variable region comprising SEQ ID NO: 14; viii. A heavy chain variable region comprising SEQ ID NO: 15; and a light chain variable region comprising SEQ ID NO: 16; ix. A heavy chain variable region comprising SEQ ID NO: 48; and a light chain variable region comprising SEQ ID NO: 8; x. A heavy chain variable region comprising SEQ ID NO: 52; and a light chain variable region comprising SEQ ID NO: 8; xi. A heavy chain variable region comprising SEQ ID NO: 56; and a light chain variable region comprising SEQ ID NO: 8; xii. A heavy chain variable region comprising SEQ ID NO: 60; and a light chain variable region comprising SEQ ID NO:8; xiii. a heavy chain variable region comprising SEQ ID NO:64; and a light chain variable region comprising SEQ ID NO:8; xiv. a heavy chain variable region comprising SEQ ID NO:68; and a light chain variable region comprising SEQ ID NO:8; xv. a heavy chain variable region comprising SEQ ID NO:72; and a light chain variable region comprising SEQ ID NO:8; xvi. a heavy chain variable region comprising SEQ ID NO:76; and a light chain variable region comprising SEQ ID NO:8; or xvii. a heavy chain variable region comprising SEQ ID NO:80; and a light chain variable region comprising SEQ ID NO:

8.

20. The method according to claim 18 or 19, wherein the cancer comprises solid tumor cell carcinoma.

21. The method according to any one of claims 18 - 20, wherein the cancer comprises liver cancer.

22. A method for inhibiting the proliferation of cells expressing glypican - 3 protein, the method comprising contacting the cells with an effective amount of an antibody or an antigen - binding fragment thereof that binds to glypican - 3 (GPC3) protein, the antibody or its antigen - binding fragment comprising: i. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:1; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4; iii. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:6; iv. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:7; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; v. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:9; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:10; vi. a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:11; and a light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:12; vii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:13; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:14; viii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:15; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:48; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; x. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:52; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:56; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:60; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:64; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:68; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xv. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:72; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; xvi. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:76; and The light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; Or xvii. The heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:80; And The light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

23. The method according to claim 22, wherein the antibody or antigen-binding fragment thereof of claim 1 comprises: i. A heavy chain variable region comprising SEQ ID NO:1; and A light chain variable region comprising SEQ ID NO:2; ii. A heavy chain variable region comprising SEQ ID NO:3; and A light chain variable region comprising SEQ ID NO:4; iii. A heavy chain variable region comprising SEQ ID NO:5; and A light chain variable region comprising SEQ ID NO:6; iv. A heavy chain variable region comprising SEQ ID NO:7; and A light chain variable region comprising SEQ ID NO:8; v. A heavy chain variable region comprising SEQ ID NO:9; and A light chain variable region comprising SEQ ID NO:10; vi. A heavy chain variable region comprising SEQ ID NO:11; and A light chain variable region comprising SEQ ID NO:12; vii. A heavy chain variable region comprising SEQ ID NO:13; and A light chain variable region comprising SEQ ID NO:14; viii. A heavy chain variable region comprising SEQ ID NO:15; and A light chain variable region comprising SEQ ID NO:16; ix. A heavy chain variable region comprising SEQ ID NO:48; and A light chain variable region comprising SEQ ID NO:8; x. A heavy chain variable region comprising SEQ ID NO:52; and A light chain variable region comprising SEQ ID NO:8; xi. A heavy chain variable region comprising SEQ ID NO:56; and A light chain variable region comprising SEQ ID NO:8; xii. A heavy chain variable region comprising SEQ ID NO:60; and A light chain variable region comprising SEQ ID NO:8; xiii. A heavy chain variable region comprising SEQ ID NO:64; and A light chain variable region comprising SEQ ID NO:8; xiv. A heavy chain variable region comprising SEQ ID NO:68; and A light chain variable region comprising SEQ ID NO:8; xv. A heavy chain variable region comprising SEQ ID NO:72; and A light chain variable region comprising SEQ ID NO:8; xvi. A heavy chain variable region comprising SEQ ID NO:76; and A light chain variable region comprising SEQ ID NO:8; or xvii. A heavy chain variable region comprising SEQ ID NO:80; and A light chain variable region comprising SEQ ID NO:

8.

24. The method according to claim 22 or 23, wherein the cell comprises a cancer cell.

25. The method according to any one of claim 24, wherein the cell comprises a liver cancer cell.

26. A method for detecting the presence of glypican-3 (GPC3) protein in a biological sample, comprising contacting the biological sample with an effective amount of an antibody or an antigen-binding fragment thereof that binds to glypican-3 (GPC3) protein, wherein the contacting is carried out under conditions that allow the antibody or its antigen-binding fragment to form a complex with GPC3 present in the sample, and measuring a detectable signal associated with the formation of the complex, wherein the antibody or its antigen-binding fragment comprises: i. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 1; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:3; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 4; iii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 5; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 6; iv. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 7; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 8; v. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 9; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 10; vi. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 12; vii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 13; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14; viii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:48; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; x. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:52; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:56; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:60; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:64; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:68; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:72; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xvi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:76; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; or xvii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:80; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

27. The method according to claim 26, wherein the antibody or antigen-binding fragment thereof of claim 1 comprises: i. a heavy chain variable region comprising SEQ ID NO:1; and a light chain variable region comprising SEQ ID NO:2; ii. a heavy chain variable region comprising SEQ ID NO:3; and a light chain variable region comprising SEQ ID NO:4; iii. a heavy chain variable region comprising SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:6; iv. a heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:8; v. a heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:10; vi. a heavy chain variable region comprising SEQ ID NO:11; and a light chain variable region comprising SEQ ID NO:12; vii. a heavy chain variable region comprising SEQ ID NO:13; and a light chain variable region comprising SEQ ID NO:14; viii. a heavy chain variable region comprising SEQ ID NO:15; and a light chain variable region comprising SEQ ID NO:16; ix. a heavy chain variable region comprising SEQ ID NO:48; and a light chain variable region comprising SEQ ID NO:8; x. a heavy chain variable region comprising SEQ ID NO:52; and a light chain variable region comprising SEQ ID NO:8; xi. a heavy chain variable region comprising SEQ ID NO:56; and a light chain variable region comprising SEQ ID NO:8; xii. a heavy chain variable region comprising SEQ ID NO:60; and a light chain variable region comprising SEQ ID NO:8; xiii. a heavy chain variable region comprising SEQ ID NO:64; and a light chain variable region comprising SEQ ID NO:8; xiv. a heavy chain variable region comprising SEQ ID NO:68; and a light chain variable region comprising SEQ ID NO:8; xv. a heavy chain variable region comprising SEQ ID NO:72; and a light chain variable region comprising SEQ ID NO:8; xvi. a heavy chain variable region comprising SEQ ID NO:76; and a light chain variable region comprising SEQ ID NO:8; or xvii. a heavy chain variable region comprising SEQ ID NO:80; and a light chain variable region comprising SEQ ID NO:

8.

28. The method according to claim 26 or 27, wherein the antibody or antigen-binding fragment thereof comprises a detectable label.

29. The method according to any one of claims 18 - 21, wherein the subject is a human.

30. The method according to any one of claims 22 - 25, wherein the cell is a human cell.

31. The method according to any one of claims 26 - 28, wherein the biological sample is from a human.

32. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof that binds to glypican-3 (GPC3) protein, wherein the antibody or the antigen-binding fragment thereof comprises: i. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:1; and A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:3; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4; iii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:6; iv. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:7; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; v. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:9; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:10; vi. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:11; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:12; vii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:13; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:14; viii. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:15; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. A heavy chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:48; And A light chain variable region comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:8; x. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:52; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:56; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:60; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:64; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:68; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:72; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xvi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:76; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; Or xvii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:80; And A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

33. The isolated polynucleotide according to claim 32, wherein the antibody or its antigen-binding fragment comprises: i. A heavy chain variable region comprising SEQ ID NO:1; and A light chain variable region comprising SEQ ID NO:2; ii. A heavy chain variable region comprising SEQ ID NO:3; and A light chain variable region comprising SEQ ID NO:4; iii. A heavy chain variable region comprising SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:6; iv. a heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:8; v. a heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:10; vi. a heavy chain variable region comprising SEQ ID NO:11; and a light chain variable region comprising SEQ ID NO:12; vii. a heavy chain variable region comprising SEQ ID NO:13; and a light chain variable region comprising SEQ ID NO:14; viii. a heavy chain variable region comprising SEQ ID NO:15; and a light chain variable region comprising SEQ ID NO:16; ix. a heavy chain variable region comprising SEQ ID NO:48; and a light chain variable region comprising SEQ ID NO:8; x. a heavy chain variable region comprising SEQ ID NO:52; and a light chain variable region comprising SEQ ID NO:8; xi. a heavy chain variable region comprising SEQ ID NO:56; and a light chain variable region comprising SEQ ID NO:8; xii. a heavy chain variable region comprising SEQ ID NO:60; and a light chain variable region comprising SEQ ID NO:8; xiii. a heavy chain variable region comprising SEQ ID NO:64; and a light chain variable region comprising SEQ ID NO:8; xiv. a heavy chain variable region comprising SEQ ID NO:68; and a light chain variable region comprising SEQ ID NO:8; xv. a heavy chain variable region comprising SEQ ID NO:72; and a light chain variable region comprising SEQ ID NO:8; xvi. a heavy chain variable region comprising SEQ ID NO:76; and a light chain variable region comprising SEQ ID NO:8; or xvii. a heavy chain variable region comprising SEQ ID NO:80; and a light chain variable region comprising SEQ ID NO:

8.

34. A isolated recombinant cell that produces an antibody or an antigen-binding fragment thereof that binds to the glypican 3 (GPC3) protein, wherein the antibody or the antigen-binding fragment thereof comprises: i. a heavy chain variable region that comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:1; and a light chain variable region that comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2; ii. The heavy chain variable region, which comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 3; and a light chain variable region that comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4; iii. a heavy chain variable region that comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5; and a light chain variable region that comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:6; iv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:7; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; v. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:9; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:10; vi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:11; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:12; vii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:13; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:14; viii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:15; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:16; ix. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:48; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; x. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:52; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:56; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:60; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:64; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xiv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:68; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xv. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:72; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; xvi. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:76; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:8; or xvii. A heavy chain variable region, said heavy chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:80; and A light chain variable region, said light chain variable region comprising: CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:

8.

35. The isolated polynucleotide according to claim 34, wherein the antibody or antigen-binding fragment thereof comprises: i. A heavy chain variable region comprising SEQ ID NO:1; and A light chain variable region comprising SEQ ID NO:2; ii. A heavy chain variable region comprising SEQ ID NO:3; and A light chain variable region comprising SEQ ID NO:4; iii. A heavy chain variable region comprising SEQ ID NO:5; and A light chain variable region comprising SEQ ID NO:6; iv. A heavy chain variable region comprising SEQ ID NO:7; and A light chain variable region comprising SEQ ID NO:8; v. A heavy chain variable region comprising SEQ ID NO:9; and A light chain variable region comprising SEQ ID NO:10; vi. A heavy chain variable region comprising SEQ ID NO:11; and A light chain variable region comprising SEQ ID NO:12; vii. A heavy chain variable region comprising SEQ ID NO:13; and A light chain variable region comprising SEQ ID NO:14; viii. A heavy chain variable region comprising SEQ ID NO:15; and A light chain variable region comprising SEQ ID NO:16; ix. A heavy chain variable region comprising SEQ ID NO:48; and A light chain variable region comprising SEQ ID NO:8; x. A heavy chain variable region comprising SEQ ID NO:52; and A light chain variable region comprising SEQ ID NO:8; xi. A heavy chain variable region comprising SEQ ID NO:56; and The light chain variable region comprising SEQ ID NO:8; xii. The heavy chain variable region comprising SEQ ID NO:60; and The light chain variable region comprising SEQ ID NO:8; xiii. The heavy chain variable region comprising SEQ ID NO:64; and The light chain variable region comprising SEQ ID NO:8; xiv. The heavy chain variable region comprising SEQ ID NO:68; and The light chain variable region comprising SEQ ID NO:8; xv. The heavy chain variable region comprising SEQ ID NO:72; and The light chain variable region comprising SEQ ID NO:8; xvi. The heavy chain variable region comprising SEQ ID NO:76; and The light chain variable region comprising SEQ ID NO:8; or xvii. The heavy chain variable region comprising SEQ ID NO:80; and The light chain variable region comprising SEQ ID NO:8.

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