GPC3 binding agents, conjugates thereof and methods of use thereof

Abstract

The present invention provides anti-GPC3 antibodies, their antigen-binding portions and their GPC3 conjugates for use in the treatment of cancer.The present disclosure in part provides variant ARD103 glypican-3 (GPC3) binding antibodies, their antigen-binding portions and related binding agents that specifically bind to GPC3, and their conjugates, which show improved therapeutic properties.GPC3 is an important and advantageous therapeutic target for the treatment of certain cancers.GPC3 binding antibodies, their antigen-binding portions and their binding agents and conjugates provide compositions and methods based on the use of such antibodies, antigen-binding portions and related binding agents, and their conjugates in the treatment of GPC3+ cancers.

Description

[Technical field]

[0001] Description of sequence listing The contents of the electronic sequence listing (120301_404WO_seqListing_FINAL.xml; size: 161 KB; creation date: November 18, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0002] background Effective, tumor-targeted treatments for various types of cancer remain a critical need to improve patient survival. There were 905,700 cases of hepatocellular carcinoma worldwide in 2020, with 830,200 deaths. In the same year, lung and gastric cancer accounted for 1.8 million deaths and 770,000 deaths, respectively. Currently, there are few treatment options for hepatocellular carcinoma. Glypican-3, or GPC3, is known to be overexpressed on human malignant cells and highly expressed in these tumors, as well as colorectal, lung, esophageal, cervical, head and neck, breast cancer, including triple-negative breast cancer, ovarian, renal cell, germ cell (testicular) cancer, vulvar cancer, melanoma, gastric, sarcoma, and bladder cancer. Because of the limited expression of GPC3 in normal adult tissues, targeting GPC3 using antibodies armed with cytotoxic agents (antibody-drug conjugates) offers a method to selectively attack cancer cells and spare normal tissues. Summary of the Invention [Means for solving the problem]

[0003] A brief summary The present disclosure provides, in part, variant ARD103 glypican-3 (GPC3) binding antibodies, their antigen-binding portions, and related binding agents that specifically bind to GPC3, as well as conjugates thereof, which exhibit improved therapeutic properties.GPC3 is an important and advantageous therapeutic target for the treatment of certain cancers.GPC3 binding antibodies, their antigen-binding portions, and their binding agents and conjugates provide compositions and methods based on the use of such antibodies, antigen-binding portions, and related binding agents, as well as conjugates thereof, in the treatment of GPC3+ cancers.Thus, the present invention provides methods, compositions, kits, and products related to variant ARD103 GPC3 antibodies, antigen-binding portions, binding agents, and conjugates. In some embodiments a conjugate is provided, the conjugate comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 A binder comprising: the heavy and light chain framework regions are optionally modified with 1 to 8 amino acid substitutions, deletions or insertions in the framework regions; The binding agent specifically binds to human GPC3; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; Includes.

[0004] In some embodiments, the binder is (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Includes.

[0005] In some embodiments a conjugate is provided, the conjugate comprising: A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:17, each disposed within a light chain framework region; (ii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:23, each disposed within a light chain framework region; (iii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:27, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:28, each disposed within a light chain framework region; (iv) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:104, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:32, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:33, each disposed within a light chain framework region; (v) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:4, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:36, each disposed within a light chain framework region; (vi) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:41, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:42, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:43, each disposed within a light chain framework region; (vii) the VH region comprises a complementarity determining region HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:48, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises a LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:49, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:50, each disposed within a light chain framework region; or (viii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 55, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 6, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56, each disposed within a light chain framework region. with a binder; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; Includes.

[0006] In some embodiments, the framework regions are murine framework regions.

[0007] In some embodiments, the framework regions are human framework regions.

[0008] In some embodiments, the binding agent is an antibody or an antigen-binding portion thereof.

[0009] In some embodiments, the binding agent is a monoclonal antibody, a Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0010] In some embodiments, the heavy chain variable region further comprises a heavy chain constant region.

[0011] In some embodiments, the heavy chain constant region is of the human IgG isotype.

[0012] In some embodiments, the heavy chain constant region is an IgG1 constant region.

[0013] In some embodiments, the IgG1 heavy chain constant region has the amino acid sequence set forth in SEQ ID NO:57 or 59.

[0014] In some embodiments, the heavy chain constant region is an IgG4 constant region.

[0015] In some embodiments, the light chain variable region further comprises a light chain constant region.

[0016] In some embodiments, the light chain constant region is of the kappa isotype.

[0017] In some embodiments, the kappa light chain constant region has the amino acid sequence set forth in SEQ ID NO:61.

[0018] In some embodiments of the binder of the present disclosure, (i) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 65 or 66, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 67; (ii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 70; (iii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 71; (iv) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 130 or 131, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 74; (v) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 72 or 73, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 75; (vi) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 76 or 77, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 78; (vii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 79 or 80, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 81; or (viii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 82 or 83, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 84.

[0019] In some embodiments, the linker is attached to the binder via an interchain disulfide residue, an engineered cysteine, a glycan or modified glycan, the N-terminal residue of the binder, or a polyhistidine residue attached to the binder.

[0020] In some embodiments, the average drug loading of the conjugate is about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

[0021] In some embodiments, the binding agent is monospecific.

[0022] In some embodiments, the binding agent is bivalent.

[0023] In some embodiments, the binding agent comprises a second binding domain and the binding agent is bispecific.

[0024] In some embodiments, the cytotoxic agent is selected from the group consisting of an auristatin, a camptothecin, a duocarmycin, and a calicheamicin.

[0025] In some embodiments, the cytotoxic agent is an auristatin.

[0026] In some embodiments, the cytotoxic agent is monomethylauristatin E (MMAE).

[0027] In some embodiments, the cytotoxic agent is a camptothecin.

[0028] In some embodiments, the cytotoxic agent is exatecan.

[0029] In some embodiments, the cytotoxic agent is a calicheamicin.

[0030] In some embodiments, the cytotoxic agent is SN-38 (also known as 7-ethyl-10-hydroxycamptothecin).

[0031] In some embodiments, the linker is selected from mc-VC-PAB, CL2, CL2A, and (succinimid-3-yl-N)-(CH2). n 2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (SEQ ID NO: 96), 2 represents an integer of 2 to 8).

[0032] In some embodiments, the linker is mc-VC-PAB.

[0033] In some embodiments, the linker is attached to at least one molecule of MMAE.

[0034] In some embodiments, the linker is CL2A.

[0035] In some embodiments, it is linked to at least one molecule of SN-38.

[0036] In some embodiments, the linker is CL2.

[0037] In some embodiments, it is linked to at least one molecule of SN-38.

[0038] In some embodiments, the linker is (succinimid-3-yl-N)-(CH) n 2 -C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (SEQ ID NO: 96), 2 represents an integer from 2 to 8).

[0039] In some embodiments, the linker is attached to at least one molecule of exatecan.

[0040] In some embodiments, a pharmaceutical composition is provided comprising a conjugate of any of the embodiments described herein and a pharma- ceutically acceptable carrier.

[0041] In some embodiments, a nucleic acid encoding a binding agent of any of the embodiments described herein is provided.

[0042] In some embodiments, a vector is provided that includes the nucleic acid of the preceding embodiments.

[0043] In some embodiments, a cell line is provided that comprises a nucleic acid of any of the embodiments described herein.

[0044] In some embodiments, methods of treating GPC3+ cancer are provided, comprising administering to a subject in need of treatment a therapeutically effective amount of any of the conjugate embodiments of the conjugates described herein or a pharmaceutical composition of any of these conjugates.

[0045] In some embodiments of the method, the GPC3+ cancer is a cancer or malignant tumor.

[0046] In some embodiments of the method, the GPC3+ cancer is selected from hepatocellular carcinoma, lung cancer, such as small cell lung cancer, squamous cell lung cancer, and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, renal cell carcinoma, breast cancer (e.g., triple-negative breast cancer), melanoma, germ cell cancer (e.g., testicular), vulvar cancer, gastric cancer, sarcoma, and bladder cancer.

[0047] In some embodiments of the method, it further comprises administering an immunotherapy to the subject.

[0048] In some embodiments of the methods, the immunotherapy comprises an immune checkpoint inhibitor.

[0049] In some embodiments of the methods, the immune checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

[0050] In some embodiments of the method, the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0051] In some embodiments, the method further comprises administering chemotherapy to the subject.

[0052] In some embodiments of the method, the conjugate is administered intravenously.

[0053] In some embodiments of the method, the conjugate is administered at a dose of about 0.1 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 12 mg / kg.

[0054] In some embodiments, a method is provided for improving the treatment outcome of a subject undergoing immunotherapy and / or chemotherapy for a GPC3+ cancer, comprising administering an effective amount of the immunotherapy or chemotherapy to a subject having cancer and administering to the subject a therapeutically effective amount of a conjugate of any of the conjugate embodiments described herein or a pharmaceutical composition of any of the conjugates described herein, wherein the treatment outcome of the subject is improved compared to administration of the immunotherapy or chemotherapy alone.

[0055] In some embodiments, the improved treatment outcome is an objective response selected from stable disease, a partial response, or a complete response.

[0056] In some embodiments, the improved treatment outcome is a reduction in tumor burden.

[0057] In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0058] In some embodiments, the immunotherapy is an immune checkpoint inhibitor.

[0059] In some embodiments, the immune checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4.

[0060] In some embodiments, the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0061] In some embodiments, the conjugate is administered intravenously.

[0062] In some embodiments, the conjugate is administered at a dose of about 0.1 mg / kg to about 10 mg / kg.

[0063] In some embodiments, there is provided a use of a conjugate described herein or a pharmaceutical composition of a conjugate described herein for treating a GPC3+ cancer in a subject.

[0064] In some embodiments, there is provided a use of a conjugate as described herein, or a pharmaceutical composition of any of the conjugates described herein, for treating GPC3+ cancer in a subject undergoing immunotherapy or chemotherapy.

[0065] These and other aspects of the present disclosure may be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief description of the drawings]

[0066] [Figure 1] FIG. 1 shows a graph of the effect of heat treatment on binding of anti-GPC3 scFv to recombinant human GPC3.

[0067] [Diagram 2] FIG. 2 shows a graph of binding of the lead IgG variant of ARD-103 to recombinant hGPC3 as determined by ELISA.

[0068] [Diagram 3] FIG. 3 shows a graph depicting the in vitro cytotoxic activity of the MMAE conjugate of the lead variant of ARD-103 against GPC3+HepG2 liver cancer cells and a table of IC50 values ​​for the MMAE conjugate of the lead variant of ARD-103.

[0069] [Figure 4] FIG. 4 shows a graph of the antitumor efficacy of ARD103-CL2A-SN38 and CL2A-SN38 conjugates of three mAb variants in a HepG2-C3A liver cancer mouse xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description The present disclosure provides anti-GPC3 antibodies, cytotoxic agent conjugates comprising anti-GPC3 antibodies, and pharmaceutical compositions comprising such antibodies and conjugates.The antibodies, conjugates and pharmaceutical compositions of the present disclosure are useful for treating GPC3+ cancer, alone or in combination with other cancer therapeutics.The anti-GPC3 antibodies of the present disclosure show enhanced binding ability to GPC3 after heat treatment compared to reference ARD103 antibodies.The conjugates of the anti-GPC3 antibodies to MMAE of the present disclosure show the same in vitro cytotoxicity against hepatocellular carcinoma cells as the reference ARD103 antibody conjugates.

[0071] For convenience, certain terms in the specification, examples and claims are defined here. Unless otherwise stated or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to help explain certain embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0072] As used herein, unless otherwise indicated, the terms "a" and "an" are intended to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms as used herein shall include the plural and plural terms shall include the singular.

[0073] The use of the alternative (e.g., "or") should be understood to mean either, both, or any combination thereof of the alternatives. As used throughout this disclosure, the terms "include" and "comprise" are used interchangeably.

[0074] "Optionally" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances when the element, component, event, or circumstance occurs and instances when it does not occur.

[0075] The phrase "at least one of" when followed by a list of items or elements refers to an open-ended collection of one or more elements in the list, which may, but does not necessarily, contain multiple elements.

[0076] The term "about" as used throughout this disclosure in the context of a number refers to a range centered around that number, 15% less than that number, and 15% more than that number. The term "about" as used in the context of a range refers to an expanded range that is 15% less than the minimum number recited in the range and 15% more than the maximum number recited in the range.

[0077] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range should be understood to include any value (including integers or fractions) or subrange within the recited range, unless otherwise stated.

[0078] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense, i.e., "without being limited to", rather than an exclusive or exhaustive sense.

[0079] The terms "reduce," "reduce," "reduced," "reduction," "reduce," and "inhibit" are all used generally herein to mean a statistically significant decrease compared to a reference.

[0080] The terms "increased," "increase" or "enhance" or "activate" are all used herein to generally mean a statically significant amount of increase relative to a reference.

[0081] As used herein, the term "isolated" or "partially purified" refers to a nucleic acid, polypeptide or protein that has been separated from at least one other component (e.g., a nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide or protein found in its natural source and / or that would be present with the nucleic acid, polypeptide or protein when expressed by a cell or would be secreted in the case of secreted polypeptides and proteins. Chemically synthesized nucleic acids, polypeptides or proteins, or those synthesized using in vitro transcription / translation, are considered "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid, polypeptide or protein that is at least 95% by weight of the nucleic acid, polypeptide or protein of interest, e.g., at least 96%, at least 97%, at least 98%, at least 99% or more.

[0082] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent residues, respectively. The terms "protein" and "polypeptide" also refer to a polymer of proteinaceous amino acids, including modified amino acids (e.g., phosphorylation, glycation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments and analogs of the foregoing.

[0083] GPC3 or glypican-3 is a glycosylphosphatidylinositol anchored cell surface protein that can function as a cell adhesion protein. (Also called DGSX, OCI-5, SDYS, SGB, SGBS.) It has been reported to be overexpressed in hepatocellular carcinoma, lung cancer such as small cell lung cancer and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, breast cancer, renal cell carcinoma, gastric cancer, sarcoma and bladder cancer, among other cancers. GPC3 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NCBI Ref Seq.NP_001158089.1 (SEQ ID NO: 85), NP_001158090.1 (SEQ ID NO: 86), NP_001158091.1 (SEQ ID NO: 87) and NP_004475.1 (SEQ ID NO: 88), which sequences are incorporated herein by reference.

[0084] As used herein, "epitope" typically refers to amino acids bound by an immunoglobulin VH / VL pair, such as the antibodies and binding agents described herein. Epitopes can be formed on a polypeptide from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An epitope defines the minimal binding site of an antibody or other binding agent and thus represents the target of specificity of the antibody, its antigen-binding portion, or other immunoglobulin-based binding agent. In the case of a single domain antibody, the epitope represents the structural unit to which the variable domain binds alone.

[0085] As used herein, "specifically binds" refers to a binding agent (e.g., an antibody or antigen-binding portion thereof) described herein that specifically binds to an antigen of interest within 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Specific binding refers to the ability to bind to a target such as GPC3 with a KD of 100 M or less. Specific binding can be influenced, for example, by the affinity and avidity of the antibody or other binding agent and the concentration of the target polypeptide. Those skilled in the art can use any suitable method, such as titrating the binding agent in a suitable cell binding assay, to determine the appropriate conditions under which the antibodies and other binding agents described herein selectively bind to GPC3. A binding agent that specifically binds to GPC3 is not displaced by a dissimilar competitor. In certain embodiments, an anti-GPC3 antibody or antigen-binding portion thereof is said to specifically bind to GPC3 when it preferentially recognizes its target antigen GPC3 in a complex mixture of proteins and / or macromolecules.

[0086] In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein is administered in a dose of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less dissociation constant (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -5 M~10 -6 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -6 M~10 -7 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -7 M~10 -8 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -8 M~10 -9 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -9 M~10 -10 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -10 M~10 -11 Dissociation constant of M (K DIn some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at about 10 -11 M~10 -12 Dissociation constant of M (K D In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a GPC3 polypeptide at 10 -12 Dissociation constant (K D ) specifically binds to GPC3 polypeptide.

[0087] As used throughout this disclosure, "identical" or "identity" refers to the similarity between a DNA, RNA, nucleotide, amino acid or protein sequence and another DNA, RNA, nucleotide, amino acid or protein sequence. Identity can be expressed in terms of the percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequence identity to a reference DNA sequence may be the percentage of DNA nucleotides in a candidate sequence that are identical to the DNA nucleotides in the reference DNA sequence after aligning the sequences. Percent (%) sequence identity to a reference amino acid sequence may be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. As used throughout this disclosure, percent sequence identity values ​​are generated using the NCBI BLAST 2.0 software as defined by Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 2007, 25, 3389-3402, with parameters set to default values.

[0088] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment.

[0089] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0090] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," which when used in connection with percentages can mean + / - 1%.

[0091] The terms "statistically significant" or "significant" refer to statistical significance, generally meaning a difference of two standard deviations (2 SD) above or below a reference value.

[0092] Other terms are defined herein within the description of various aspects of the disclosure.

[0093] I. Antibodies Provided herein are variant ARD103 binding antibodies (also referred to as anti-GPC3 antibodies or GPC3 binding antibodies) and antigen-binding portions thereof that specifically bind to glypican-3 (GPC3).Also provided herein are ARD103 (anti-GPC3) binding antibodies and antigen-binding portions and conjugates of cytotoxic agents (also referred to as GPC3 conjugates).In some embodiments, the GPC3 conjugates reduce the number of GPC3+ cancer cells in a subject.

[0094] In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof is (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Includes.

[0095] In some embodiments, the anti-GPC3 binding antibody or antigen-binding portion thereof is (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Including, The heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0096] In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof is (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Including, The heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable regions are unmodified.

[0097] In some embodiments, disclosed herein is a binding agent, the binding agent comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 wherein the binding agent specifically binds to GPC3.

[0098] In some embodiments, disclosed herein is a binding agent, the binding agent comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Including, The heavy and light chain variable framework regions are optionally modified with 1-8, 1-6, 1-4 or 1-2 conservative amino acid substitutions in the framework regions, the CDRs of the heavy or light chain variable regions are unmodified, and the binding agent specifically binds to GPC3.

[0099] In some embodiments, disclosed herein is a binding agent, the binding agent comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 Including, The heavy and light chain variable framework regions are optionally modified with 1-8, 1-6, 1-4 or 1-2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. As described herein, the binding agent comprises an anti-GPC3 antibody or an antigen-binding portion thereof, and may comprise other peptides or polypeptides covalently bound to the anti-GPC3 antibody or an antigen-binding portion thereof. In any of these embodiments, the binding agent specifically binds to GPC3.

[0100] In some embodiments, the CDRs of the heavy and / or light chains of an antibody or antigen-binding fragment thereof may be identified by using any one of the following methods: Kabat, Chothia, AbM, Contact, IMGT, and / or Aho. In some embodiments, the CDRs are defined by Kabat.

[0101] In some embodiments, provided is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:17, each disposed within a light chain framework region; (ii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:23, each disposed within a light chain framework region; (iii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:27, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:28, each disposed within a light chain framework region; (iv) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:104, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:32, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:33, each disposed within a light chain framework region; (v) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:4, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:36, each disposed within a light chain framework region; (vi) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:41, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:42, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:43, each disposed within a light chain framework region; (vii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:48, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:49, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:50, each disposed within a light chain framework region; or (viii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 55, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 6, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56, each disposed within a light chain framework region; Each VH and VL comprises a humanized framework region, and the binding agent specifically binds to GPC3.

[0102] In some embodiments, the compositions and methods described herein relate to the reduction of GPC3+ cells in a subject (e.g., the reduction of the number of GPC3+ cells in a cancer or tumor) by anti-GPC3 antibodies, their antigen-binding portions, other binding agents or conjugates thereof in vivo. In some embodiments, the compositions and methods described herein relate to the treatment of GPC3+ cancer in a subject by administering anti-GPC3 antibodies, their antigen-binding portions, other binding agents or conjugates thereof.

[0103] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds to an antigen. The term generally refers to antibodies that are composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions that comprise a full-length antibody (having heavy and light chain constant regions) and its antigen-binding portion, e.g., intact monoclonal antibodies, Fab, Fab', F(ab') 2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAbs), diabodies, multispecific antibodies, dual specific antibodies, bispecific antibodies, and single chain antibodies (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). Antibodies can include, for example, polyclonal, monoclonal, and genetically engineered antibodies, as well as antigen-binding fragments thereof. Antibodies can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody.

[0104] Each heavy chain typically consists of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains CH1, CH2 and CH3, and optionally a fourth domain CH4. Each light chain typically consists of a variable region (abbreviated as VL) and a constant region. The light chain constant region is a CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity determining regions (CDRs), and may be interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. This structure is known to those skilled in the art. The CDR and FR sequences may be determined by several different numbering schemes, including Kabat, Chothia, AbM, Contact, IMGT and / or Aho. In some embodiments, the CDRs and FRs are defined by Kabat.

[0105] In some embodiments, the antigen-binding portion comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3).

[0106] The amino acid sequences of the VH CDRs and VL CDRs, VH and VL, and constant regions of exemplary anti-GPC3 antibodies of the present disclosure are shown in Table 1. The phrase "unmodified CDRs of the heavy chain variable region or light chain variable region" refers to these VH and VL CDRs that do not have amino acid substitutions, deletions, or insertions (e.g., SEQ ID NOs: 3, 15, 5, 16, 7, and 17; SEQ ID NOs: 3, 22, 5, 16, 7, and 23; SEQ ID NOs: 3, 22, 5, 27, 7, and 28; SEQ ID NOs: 3, 104, 5, 32, 7, and 33; SEQ ID NOs: 3, 4, 5, 16, 7, and 36; SEQ ID NOs: 3, 41, 5, 42, 7, and 43; SEQ ID NOs: 3, 48, 5, 49, 7, and 50; or SEQ ID NOs: 3, 55, 5, 6, 7, and 56). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0107] As used herein, the "antigen-binding portion" or "antigen-binding fragment" of an anti-GPC3 antibody refers to a region of an antibody molecule that specifically binds to an antigen. In some embodiments, the antigen-binding portion refers to a portion of an anti-GPC3 antibody described herein having the VH and VL sequences of the anti-GPC3 antibody (e.g., as shown in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, optionally modified as described herein). According to the term "antigen-binding portion" of an antibody, examples of antigen-binding portions include Fab, Fab', F(ab') 2, Fv, disulfide-linked Fv, scFv, single domain antibody (dAb), diabody, heavy chain antibody (hcAb), VHH, VNAR, nanobody, and single chain antibody. As used herein, the terms Fab, F(ab')2 and Fv refer to the following: (i) Fab fragment, i.e., a monovalent fragment composed of VL, VH, CL and CH1 domains, (ii) F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked together in the hinge region via a disulfide bridge, and (iii) Fv fragment composed of the VL and VH domains of an anti-GPC3 antibody. The two domains of the Fv fragment, VL and VH, are encoded by separate coding regions, but they may be further linked to each other using a synthetic linker, such as a poly G4S amino acid sequence ("(G4S)n", where n=1-5, as disclosed in SEQ ID NOs: 89, 90, 91, 92 and 93, respectively), allowing the VL and VH regions to be prepared as a single protein chain that combines to form a monovalent molecule (known as single-chain Fv (ScFv)). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as "diabodies", are included here as well. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the VH and VL domains are paired with complementary domains (VL and VH, respectively) on different chains to form two antigen-binding sites, using a linker connecting the VH and VL domains that is too short to allow the two domains to combine on the same chain (e.g., Holliger, R et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123).

[0108] Immunoglobulin constant region refers to heavy or light chain constant region. The constant region provides the general framework of the antibody and may not be directly involved in binding the antibody to the antigen, but may be involved in various effector functions such as antibody-dependent cellular cytotoxicity (ADCC), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), longer in vivo half-life compared to polypeptides lacking the Fc region, protein A binding, and possibly involvement of the antibody in transplacental transfer (see Capon et al., Nature 337:525, 1989). As used throughout this disclosure, "Fc region" refers to the heavy chain constant region segment of the Fc fragment from an antibody ("fragment crystallizable" region or Fc region), which may include one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. In some embodiments, the Fc region comprises the CH2 and CH3 domains of an IgG, IgA or IgD antibody, or the CH3 and CH4 domains of an IgM or IgE antibody.

[0109] The amino acid sequences of the constant regions of human heavy and light chains are known in the art. The constant region may be of any suitable type, which may be selected from the classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM. Some immunoglobulin classes may be further divided into isotypes, for example, IgG1, IgG2, IgG3, IgG4, or IgA1, and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins may be α, δ, ε, γ and μ, respectively. The light chain may be any one of kappa (or κ) and lambda (or λ). There are also allotypic variants of immunoglobulin constant regions, for example, IgG1, IgG2, IgG3, and IgA heavy chains, and Ig kappa light chains.

[0110] In some embodiments, the constant region can have an IgG1 isotype. In some embodiments, the constant region can have an IgG2 isotype. In some embodiments, the constant region can have an IgG3 isotype. In some embodiments, the constant region can have an IgG4 isotype. In some embodiments, the Fc region can have a hybrid isotype that includes constant domains from two or more isotypes. In some embodiments, the immunoglobulin constant region can be an IgG1 or IgG4 constant region. In some embodiments, the constant region is an IgG1 allotype variant (e.g., G1m1 or nG1m1). An exemplary amino acid sequence of an IgG1 G1m1 allotype constant region is shown in SEQ ID NO:57. An exemplary amino acid sequence of an IgG1 nG1m1 allotype constant region is shown in SEQ ID NO:59.

[0111] In some embodiments, the anti-GPC3 antibody has an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 57 or 59. In some embodiments, the anti-GPC3 antibody has a kappa light chain constant region. In some embodiments, the kappa light chain constant region has the amino acid sequence shown in SEQ ID NO: 61.

[0112] In some embodiments, the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has an amino acid sequence as set forth in any one of SEQ ID NOs: 65, 66, 68, 69, 72, 73, 76, 77, 79, 80, 82, 83, 130 and 131. In some embodiments, the anti-GPC3 antibody light chain is of the kappa isotype and has an amino acid sequence as set forth in any one of SEQ ID NOs: 67, 70, 71, 74, 75, 78, 81 and 84.

[0113] In some embodiments of the anti-GPC3 antibodies of the present disclosure, (i) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 65 or 66, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence shown in SEQ ID NO: 67; (ii) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 70; (iii) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 71; (iv) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 130 or 131, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 74; (v) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 72 or 73, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence shown in SEQ ID NO: 75; (vi) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 76 or 77, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 78; (vii) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 79 or 80, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 81; or (viii) the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 82 or 83, and the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence shown in SEQ ID NO: 84.

[0114] Furthermore, the anti-GPC3 antibody or its antigen-binding portion may be part of a larger binder formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Related to such binders is the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM et al. (1995), Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidinyl peptides, such as hexahistidinyl tags ("hexahistidinyl tags" disclosed as SEQ ID NO: 94), to produce bivalent and biotinylated scFv molecules (Kipriyanov, SM et al. (1994) Mol. Immunol. 31:1047-1058).

[0115] With respect to the VH and VL amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions or additions (insertions) to the nucleic acid encoding VH or VL, or amino acids in a polypeptide that change a single amino acid or a small percentage of amino acids in the encoded sequence, are "conservatively modified variants", where the change results in the replacement of an amino acid with a chemically similar amino acid (conservative amino acid substitution), and the altered polypeptide retains the ability to specifically bind to GPC3.

[0116] In some embodiments, conservatively modified variants of anti-GPC3 antibodies or antigen-binding portions thereof may have changes in the framework regions (FR), i.e., other than the CDRs, e.g., conservatively modified variants of anti-GPC3 antibodies have the amino acid sequences of the VH and VL CDRs (SEQ ID NOs: 3, 15, 5, 16, 7, and 17; SEQ ID NOs: 3, 22, 5, 16, 7, and 23; SEQ ID NOs: 3, 22, 5, 27, 7, and 28; SEQ ID NOs: 3, 104, 5, 32, 7, and 33; SEQ ID NOs: 3, 4, 5, 16, 7, and 36; SEQ ID NOs: 3, 41, 5, 42, 7, and 43; SEQ ID NOs: 3, 48, 5, 49, 7, and 50; or SEQ ID NOs: 3, 55, 5, 6, 7, and 56) and at least one conservative amino acid substitution in the FR. In some embodiments, the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively) collectively have no more than 8 or 6 or 4 or 2 or 1 conservative amino acid substitutions in the FRs compared to the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively). In some embodiments, the amino acid sequences of VH and VL (shown in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively) have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the FR compared to the amino acid sequences of VH and VL (shown in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively).In further aspects of any of these embodiments, conservatively modified variants of the anti-GPC3 antibodies, antigen-binding portions thereof or other binding agents exhibit specific binding to GPC3.

[0117] For conservative amino acid substitution, a given amino acid can be replaced by a residue with similar physicochemical characteristics, for example, by replacing one aliphatic residue with another aliphatic residue (e.g., Ile, Val, Leu, or Ala for each other), or by replacing one polar residue with another polar residue (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative amino acid substitutions, for example, full-region substitutions with similar hydrophobic properties, are known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, for example, antigen binding activity and specificity of the native or reference polypeptide is retained, i.e., is retained for GPC3.

[0118] For conservative substitutions, amino acids can be grouped according to the similarity of their side chain properties (A. L. Lehninger, in Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)) into: (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0119] Alternatively, for conservative substitutions, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging members of one of these classes for another.

[0120] Particular conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0121] In some embodiments, conservatively modified variants of anti-GPC3 antibodies or antigen-binding portions thereof are preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a reference VH or VL sequence, and the VH and VL CDRs (SEQ ID NOs: 3, 15, 5, 16, 7 and 17; SEQ ID NOs: 3, 22, 5, 16, 7 and 23; SEQ ID NOs: 3, 22, 5, 27, 7 and 28; SEQ ID NOs: 3, 104, 5, 32, 7 and 33; SEQ ID NOs: 3, 4, 5, 16, 7 and 36; SEQ ID NOs: 3, 41, 5, 42, 7 and 43; SEQ ID NOs: 3, 48, 5, 49, 7 and 50; or SEQ ID NOs: 3, 55, 5, 6, 7 and 56) are unaltered. As used throughout this disclosure, "identical" or "identity" refers to the similarity between a DNA, RNA, nucleotide, amino acid or protein sequence and another DNA, RNA, nucleotide, amino acid or protein sequence. Identity can be expressed in terms of the percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequence identity to a reference DNA sequence may be the percentage of DNA nucleotides in a candidate sequence that are identical to the DNA nucleotides in the reference DNA sequence after aligning the sequences. Percent (%) sequence identity to a reference amino acid sequence may be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. As used throughout this disclosure, percent sequence identity values ​​are generated using the NCBI BLAST 2.0 software as defined by Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 2007, 25, 3389-3402, with parameters set to default values.

[0122] In some embodiments, the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively) collectively have 8 or 6 or 4 or 2 or 1 or less conservative amino acid substitutions in the framework regions compared to the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively). In some embodiments, the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively) collectively have 8 to 1, or 6 to 1, or 4 to 1, or 2 to 1 conservative amino acid substitutions in the framework regions compared to the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively). In some embodiments, the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively) collectively have 8 or 6 or 4 or 2 or 1 or less amino acid substitutions, deletions or insertions in the framework regions compared to the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively).In some embodiments, the amino acid sequences of VH and VL (set forth in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively) have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the framework regions compared to the amino acid sequences of VH and VL (set forth in SEQ ID NOs: 11 and 12, 18 and 19, 18 and 24, 128 and 29, 1 and 34, 37 and 38, 44 and 45, or 51 and 52, respectively). In some embodiments, the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively) collectively have no more than 8 or 6 or 4 or 2 or 1 amino acid substitutions, deletions or insertions compared to the VH and VL amino acid sequences (set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, respectively).

[0123] Modification of the native (or reference) amino acid sequence can be accomplished by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the desired mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the desired substitution, deletion, or insertion. The techniques for making such modifications are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 12-19, 1985); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein in their entireties.

[0124] In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof has a fully human constant region. In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof has a non-human constant region. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 65 or 66; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 68 or 69; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 68 or 69; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 71. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 130 or 131; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 74. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 72 or 73; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 75. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 76 or 77; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the anti-GPC3 antibody heavy chain is of IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 79 or 80; and / or the anti-GPC3 antibody light chain is of kappa isotype and has the amino acid sequence shown in SEQ ID NO: 81.In some embodiments, the anti-GPC3 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 82 or 83; and / or the anti-GPC3 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 84.

[0125] In various embodiments, anti-GPC3 antibodies, their antigen-binding portions and other binding agents can be produced in human, mouse or other animal derived cell lines. Recombinant DNA expression can be used to produce anti-GPC3 antibodies, their antigen-binding portions and other binding agents. This allows the production of anti-GPC3 antibodies and the generation of a spectrum of GPC3 antigen-binding portions and other binding agents (including fusion proteins) in a selected host species. Production of anti-GPC3 antibodies, their antigen-binding portions and other binding agents in bacteria, yeast, transgenic animals and chicken eggs are also alternatives to cell-based production systems. The main advantage of transgenic animals is the potential high yield from renewable resources.

[0126] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 11 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 12 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 11 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 13. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 12 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 14.

[0127] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 18 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 19 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 18 is encoded by a nucleic acid having a sequence shown in SEQ ID NO: 20. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 19 is encoded by a nucleic acid having a sequence shown in SEQ ID NO: 21.

[0128] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 18 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 24 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 18 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 25. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 24 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 26.

[0129] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 128 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 29 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 128 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 129. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 29 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 31.

[0130] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 34 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 30. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 34 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 35.

[0131] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 37 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 38 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 37 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 39. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 38 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 40.

[0132] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 44 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 45 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 44 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 46. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 45 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 47.

[0133] In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 51 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 52 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 51 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 53. In some embodiments, the anti-GPC3 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 52 is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 54.

[0134] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 65 or 66 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 67 is encoded by a nucleic acid.

[0135] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 68 or 69 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 70 is encoded by a nucleic acid.

[0136] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 68 or 69 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 71 is encoded by a nucleic acid.

[0137] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence set forth in SEQ ID NO: 130 or 131 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence set forth in SEQ ID NO: 74 is encoded by a nucleic acid.

[0138] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 72 or 73 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 75 is encoded by a nucleic acid.

[0139] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 76 or 77 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 78 is encoded by a nucleic acid.

[0140] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 79 or 80 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 81 is encoded by a nucleic acid.

[0141] In some embodiments, the anti-GPC3 heavy chain polypeptide having the amino acid sequence shown in SEQ ID NO: 82 or 83 is encoded by a nucleic acid. In some embodiments, the anti-GPC3 light chain polypeptide having the amino acid sequence shown in SEQ ID NO: 84 is encoded by a nucleic acid.

[0142] As used herein, the term "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" or "nucleotide" refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid may be either single-stranded or double-stranded. A single-stranded nucleic acid may be a single strand of a denatured double-stranded DNA. If single-stranded, the nucleic acid may be a coding strand or a non-coding (antisense) strand. A nucleic acid molecule may contain natural or non-natural subunits. A nucleic acid molecule that codes for an amino acid sequence includes all nucleotide sequences that code for the same amino acid sequence. Some versions of a nucleotide sequence may also contain introns to the extent that the introns are removed via co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may code for the same amino acid sequence as a result of redundancy or degeneracy in the genetic code or by splicing. In some embodiments, the nucleic acid may be a cDNA, e.g., a nucleic acid lacking introns.

[0143] Nucleic acid molecules encoding the amino acid sequence of anti-GPC3 antibodies, their antigen-binding portions, and other binding agents can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding anti-GPC3 antibodies, antigen-binding portions, or other binding agents. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding anti-GPC3 antibodies or antigen-binding portions, as well as other binding agents. As described herein, at least the nucleic acid sequence encoding anti-GPC3 antibodies, their antigen-binding portions, binding agents, or polypeptides thereof can be recombined with vector DNA according to conventional techniques, such as restriction enzyme digestion to provide blunt or sticky ends for ligation, suitable ends, filling sticky ends as necessary, alkaline phosphatase treatment to avoid undesired ligation, and ligation with a suitable ligase. Techniques for such manipulations are disclosed, for example, by Maniatis et al., Molecular Cloning, Lab.Manual (Cold Spring Harbor Lab.Press, NY, 1982 and 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and the techniques can be used to construct nucleic acid sequences and vectors encoding anti-GPC3 antibodies or antigen-binding portions thereof or VH or VL polypeptides thereof.

[0144] A nucleic acid molecule such as DNA contains a nucleotide sequence that includes transcriptional and translational regulatory information, and is said to be "capable of being expressed" into a polypeptide when such a sequence is "operably linked" to a nucleotide sequence that encodes a polypeptide. An operable link is one in which the regulatory DNA sequence and the DNA sequence desired to be expressed (e.g., an anti-GPC3 antibody or antigen-binding portion thereof) are linked in such a way as to allow gene expression of a recoverable amount of the polypeptide or antigen-binding portion. The exact nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the art. See, for example, Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0145] Thus, expression of the anti-GPC3 antibody or antigen-binding portion thereof described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include yeast, insect, fungal, avian and mammalian cells in vivo or in situ, or bacterial or eukaryotic hosts, including host cells of mammalian, insect, avian or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine or feline origin, although any other mammalian cells may be used. In addition, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus produced can be processed in vivo or purified and processed in vitro, allowing the synthesis of the anti-GPC3 antibody or antigen-binding portion thereof described herein with a specific amino-terminal sequence. In addition, problems associated with retention of the methionine residue from the start codon in direct yeast (or bacterial) expression can be avoided. (See, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).) Recombinant anti-GPC3 antibodies or antigen-binding portions thereof can be produced using any of a range of yeast gene expression systems incorporating promoter and terminator elements from actively expressed genes that code for glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0146] Production of anti-GPC3 antibodies or antigen-binding portions thereof in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express the polypeptide by methods known to those skilled in the art. See Ausubel et al., 1987-1993.

[0147] In some embodiments, the introduced nucleic acid sequence (encoding anti-GPC3 antibody or its antigen-binding portion or its polypeptide) is incorporated into a plasmid or viral vector that can autonomously replicate in recipient host cells. Any of a wide variety of vectors can be used for this purpose, and are known and available to those skilled in the art. For example, see Ausubel et al., 1987-1993. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the copy number of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0148] Exemplary viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated virus), coronavirus, negative-stranded RNA virus, such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive-stranded RNA virus, such as picornavirus and alphavirus, and double-stranded DNA virus, including adenovirus, herpesvirus (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus) and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian type C viruses, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, 3rd ed., BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996). In some such embodiments, the viral vector is a lentivirus vector or a gamma-retrovirus vector.

[0149] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids that can replicate in E. coli. Other gene expression elements useful for expressing DNA encoding anti-GPC3 antibody or antigen-binding portion thereof include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites, such as those derived from SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983). DNA genes encoding immunoglobulins can be expressed using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin heavy chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S globin intervening sequence, the immunoglobulin and rabbit S globin polyadenylation sites, and the SV40 polyadenylation element, as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987).

[0150] In the case of immunoglobulin encoding nucleotide sequences, the transcription promoter may be, for example, human cytomegalovirus and the promoter enhancer may be cytomegalovirus and mouse / human immunoglobulin.

[0151] In some embodiments, for expression of DNA coding regions in rodent cells, the transcription promoter may be a viral LTR sequence, and the transcription promoter enhancer may be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the above expression elements to achieve expression of proteins in mammalian cells.

[0152] Each coding region or gene fusion is then assembled or inserted into an expression vector. The anti-GPC3 variable region or antigen-binding portion thereof (e.g., VH having the amino acid sequence shown in SEQ ID NO: 11 and / or VL having the amino acid sequence shown in SEQ ID NO: 12; VH having the amino acid sequence shown in SEQ ID NO: 18 and / or VL having the amino acid sequence shown in SEQ ID NO: 19; VH having the amino acid sequence shown in SEQ ID NO: 18 and / or VL having the amino acid sequence shown in SEQ ID NO: 24; VH having the amino acid sequence shown in SEQ ID NO: 128 and / or VL having the amino acid sequence shown in SEQ ID NO: 29; VH having the amino acid sequence shown in SEQ ID NO: 1 and / or VL having the amino acid sequence shown in SEQ ID NO: 34; sequence A recipient cell capable of expressing an anti-GPC3 antibody or antibody polypeptide or an antigen-binding portion thereof (VH having the amino acid sequence shown in SEQ ID NO: 37 and / or VL having the amino acid sequence shown in SEQ ID NO: 38; VH having the amino acid sequence shown in SEQ ID NO: 44 and / or VL having the amino acid sequence shown in SEQ ID NO: 45; or VH having the amino acid sequence shown in SEQ ID NO: 51 and / or VL having the amino acid sequence shown in SEQ ID NO: 52; or a variant thereof as described herein) is transfected with a nucleotide encoding an anti-GPC3 antibody or antibody polypeptide or an antigen-binding portion thereof alone or co-transfected with a polynucleotide encoding the VH and VL chain coding regions. The transfected recipient cell is cultured under conditions that allow expression of the incorporated coding regions, and the expressed antibody chain or intact antibody or antigen-binding portion is recovered from the culture.

[0153] In some embodiments, nucleic acids containing coding regions encoding an anti-GPC3 antibody or antigen-binding portion thereof (e.g., a VH having an amino acid sequence as set forth in SEQ ID NO: 11 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 12; a VH having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 19; a VH having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 24; a VH having an amino acid sequence as set forth in SEQ ID NO: 128 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 29; a VH having an amino acid sequence as set forth in SEQ ID NO: 1 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 34; a VH having an amino acid sequence as set forth in SEQ ID NO: 37 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 38; a VH having an amino acid sequence as set forth in SEQ ID NO: 44 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 45; or a VH having an amino acid sequence as set forth in SEQ ID NO: 51 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 52; or a variant thereof as described herein) are assembled into separate expression vectors that are then used to co-transfect recipient host cells. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, with each vector having a set of coding regions. This strategy results in a vector that first directs the production of nucleotide sequences in a bacterial system and allows amplification. The DNA vector thus produced and amplified in the bacterial host is then used to co-transfect eukaryotic cells, allowing the selection of co-transfected cells with the desired transfected nucleic acid (e.g., containing the heavy and light chains of an anti-GPC3 antibody). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol.Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, fused nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0154] For transfection of the expression vector and production of anti-GPC3 antibody or antigen-binding portion thereof, the recipient cell line may be a Chinese hamster ovary cell line (e.g., DG44) or a myeloma cell. The myeloma cell can synthesize, assemble and secrete the immunoglobulins encoded by the transfected immunoglobulin genes, and has the machinery for glycosylation of the immunoglobulins. For example, in some embodiments, the recipient cell is a recombinant Ig-producing myeloma cell SP2 / 0. The SP2 / 0 cell produces only the immunoglobulins encoded by the transfected genes. The myeloma cell can be grown in culture or in the peritoneal cavity of the mouse, and the secreted immunoglobulins can be obtained from the ascites.

[0155] Anti-GPC3 antibodies or antigen-binding portions thereof (e.g., a VH having the amino acid sequence shown in SEQ ID NO: 11 and / or a VL having the amino acid sequence shown in SEQ ID NO: 12; a VH having the amino acid sequence shown in SEQ ID NO: 18 and / or a VL having the amino acid sequence shown in SEQ ID NO: 19; a VH having the amino acid sequence shown in SEQ ID NO: 18 and / or a VL having the amino acid sequence shown in SEQ ID NO: 24; a VH having the amino acid sequence shown in SEQ ID NO: 128 and / or a VL having the amino acid sequence shown in SEQ ID NO: 29; a VH having the amino acid sequence shown in SEQ ID NO: 1 and / or a VL having the amino acid sequence shown in SEQ ID NO: 34; a VH having the amino acid sequence shown in SEQ ID NO: 37 and / or a VL having the amino acid sequence shown in SEQ ID NO: 38). An expression vector encoding a VL having the amino acid sequence shown in SEQ ID NO:38; a VH having the amino acid sequence shown in SEQ ID NO:44 and / or a VL having the amino acid sequence shown in SEQ ID NO:45; or a VH having the amino acid sequence shown in SEQ ID NO:51 and / or a VL having the amino acid sequence shown in SEQ ID NO:52; or a variant thereof as described herein) can be introduced into a suitable host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment. As known to those skilled in the art, Johnston et al., 240 Science 1538 (1988).

[0156] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. There are several recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., prepolypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpellier, France, 1982).

[0157] Yeast gene expression systems can be routinely evaluated for the production, secretion levels and stability of antibodies and assembled anti-GPC3 antibodies and their antigen-binding portions. A variety of yeast gene expression systems can be utilized that incorporate promoter and termination elements from actively expressed genes that code for glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1 alpha promoter. Several approaches can be taken to evaluate the optimal expression plasmid for the expression of immunoglobulins in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, for example, US Patent Publication No. 2006 / 0270045.

[0158] Bacterial strains can also be utilized as hosts for producing the antibody molecules or antigen-binding portions thereof described herein, including E. coli K12 strains, such as E. coli W3110, Bacillus species, Enterobacteriaceae, such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vectors have a replication site and specific genes that can provide phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of anti-GPC3 antibodies and antigen-binding portions thereof in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0159] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications of immunoglobulin molecules including removal of leader peptides, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or antigen-binding portions thereof.

[0160] In addition to the cells of lymphoid origin described above, mammalian cells that may be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PERC6™ cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0161] In some embodiments, one or more anti-GPC3 antibodies or antigen-binding portions thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 11 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 12; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 19; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 24; a VH having the amino acid sequence set forth in SEQ ID NO: 128 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 29; a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 10; or a VL having the amino acid sequence set forth in SEQ ID NO: 34; a VH having the amino acid sequence set forth in SEQ ID NO: 37 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 38; a VH having the amino acid sequence set forth in SEQ ID NO: 44 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 45; or a VH having the amino acid sequence set forth in SEQ ID NO: 51 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 52; or a variant thereof as described herein) can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptide according to any suitable method.

[0162] In some embodiments, an antibody or antigen-binding portion thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO:11 and / or a VL having the amino acid sequence set forth in SEQ ID NO:12; a VH having the amino acid sequence set forth in SEQ ID NO:18 and / or a VL having the amino acid sequence set forth in SEQ ID NO:19; a VH having the amino acid sequence set forth in SEQ ID NO:18 and / or a VL having the amino acid sequence set forth in SEQ ID NO:24; a VH having the amino acid sequence set forth in SEQ ID NO:128 and / or a VL having the amino acid sequence set forth in SEQ ID NO:29; a VH having the amino acid sequence set forth in SEQ ID NO:1 and / or a VL having the amino acid sequence set forth in SEQ ID NO:34; a VH having the amino acid sequence set forth in SEQ ID NO:37 and / or a VL having the amino acid sequence set forth in SEQ ID NO:38; a VH having the amino acid sequence set forth in SEQ ID NO:44 and / or a VL having the amino acid sequence set forth in SEQ ID NO:45; or a VH having the amino acid sequence set forth in SEQ ID NO:51 and / or a VL having the amino acid sequence set forth in SEQ ID NO:52; or a variant thereof as described herein) is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0163] Many vector systems are available for the expression of VH and VL chains in mammalian cells (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 11 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 12; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 19; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 24; a VH having the amino acid sequence set forth in SEQ ID NO: 128 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 29; a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 34; a VH having the amino acid sequence set forth in SEQ ID NO: 37 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 38; a VH having the amino acid sequence set forth in SEQ ID NO: 44 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 45; or a VH having the amino acid sequence set forth in SEQ ID NO: 51 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 52; or variants thereof as described herein) (see Glover, 1985). To obtain intact antibodies, various approaches can be followed. As described above, the VH and VL chains, and optionally the associated constant regions, can be co-expressed in the same cell to achieve intracellular association and linkage of the VH and VL chains into a complete tetrameric H2L2 antibody or antigen-binding portion thereof. Co-expression can occur by using either the same or different plasmids in the same host.Nucleic acids encoding a VH chain and a VL chain or antigen-binding portions thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO:11 and / or a VL having the amino acid sequence set forth in SEQ ID NO:12; a VH having the amino acid sequence set forth in SEQ ID NO:18 and / or a VL having the amino acid sequence set forth in SEQ ID NO:19; a VH having the amino acid sequence set forth in SEQ ID NO:18 and / or a VL having the amino acid sequence set forth in SEQ ID NO:24; a VH having the amino acid sequence set forth in SEQ ID NO:128 and / or a VL having the amino acid sequence set forth in SEQ ID NO:29; a VH having the amino acid sequence set forth in SEQ ID NO:1 and / or a VL having the amino acid sequence set forth in SEQ ID NO:34; a VH having the amino acid sequence set forth in SEQ ID NO:37 and / or a VL having the amino acid sequence set forth in SEQ ID NO:38; a VH having the amino acid sequence set forth in SEQ ID NO:44 and / or a VL having the amino acid sequence set forth in SEQ ID NO:45; or a VH having the amino acid sequence set forth in SEQ ID NO:51 and / or a VL having the amino acid sequence set forth in SEQ ID NO:52; or variants thereof as described herein) can be placed on the same plasmid, which is then transfected into a cell, thereby directly selecting for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, for example the VL chain, and subsequently transfected the resulting cell line with a VH chain plasmid containing a second selectable marker.Cell lines producing antibodies, antigen-binding portions thereof via either route can be synthesized in conjunction with additional selection markers, and can be used to express peptides, VH, VL, or VH+VL chains (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 11 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 12; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 19; a VH having the amino acid sequence set forth in SEQ ID NO: 18 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 24; a VH having the amino acid sequence set forth in SEQ ID NO: 128 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 29; a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 29; a VH having the amino acid sequence set forth in SEQ ID NO: 2 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 30; VL having the amino acid sequence set forth in SEQ ID NO: 34; VH having the amino acid sequence set forth in SEQ ID NO: 37 and / or VL having the amino acid sequence set forth in SEQ ID NO: 38; VH having the amino acid sequence set forth in SEQ ID NO: 44 and / or VL having the amino acid sequence set forth in SEQ ID NO: 45; or VH having the amino acid sequence set forth in SEQ ID NO: 51 and / or VL having the amino acid sequence set forth in SEQ ID NO: 52; or variants thereof as described herein) can be transfected with a plasmid encoding additional copies of the assembled anti-GPC3 antibody or antigen-binding portion thereof to generate a cell line with enhanced properties, such as higher production of the assembled anti-GPC3 antibody or antigen-binding portion thereof or enhanced stability of the transfected cell line.

[0164] Furthermore, plants have emerged as a convenient, safe and economical alternative expression system for recombinant antibody production based on large-scale culture of microbial or animal cells. Anti-GPC3 antibodies or antigen-binding portions can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, restricted to intracellular plastids, or restricted to seeds (endosperm). See, for example, U.S. Patent Publication No. 2003 / 0167531; U.S. Patent No. 6,080,560; U.S. Patent No. 6,512,162; PCT Publication No. WO0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0165] In the case of an intact antibody, the variable regions (VH and VL) of the anti-GPC3 antibody (e.g., VH having the amino acid sequence shown in SEQ ID NO: 11 and / or VL having the amino acid sequence shown in SEQ ID NO: 12; VH having the amino acid sequence shown in SEQ ID NO: 18 and / or VL having the amino acid sequence shown in SEQ ID NO: 19; VH having the amino acid sequence shown in SEQ ID NO: 18 and / or VL having the amino acid sequence shown in SEQ ID NO: 24; VH having the amino acid sequence shown in SEQ ID NO: 128 and / or VL having the amino acid sequence shown in SEQ ID NO: 29; VH having the amino acid sequence shown in SEQ ID NO: 128 and / or VL ... H and / or VL having the amino acid sequence shown in SEQ ID NO: 34; VH having the amino acid sequence shown in SEQ ID NO: 37 and / or VL having the amino acid sequence shown in SEQ ID NO: 38; VH having the amino acid sequence shown in SEQ ID NO: 44 and / or VL having the amino acid sequence shown in SEQ ID NO: 45; or VH having the amino acid sequence shown in SEQ ID NO: 51 and / or VL having the amino acid sequence shown in SEQ ID NO: 52; or variants thereof described herein) are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. Human constant region DNA sequences can be isolated according to known procedures from various human cells, such as immortalized B cells (PCT Publication No. WO87 / 02671; incorporated herein by reference in its entirety). Anti-GPC3 antibodies can include both light chain constant regions and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and sometimes CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted.

[0166] Alternatively, techniques described for the production of single chain antibodies (e.g., U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989); which are incorporated herein by reference in their entireties) can be adapted to produce single chain antibodies that specifically bind GPC3. Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region (e.g., having the amino acid sequences set forth in SEQ ID NOs: 11 and 12, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 24, SEQ ID NOs: 128 and 29, SEQ ID NOs: 1 and 34, SEQ ID NOs: 37 and 38, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 51 and 52, or variants thereof described herein (e.g., modified with 1-8 amino acid substitutions, deletions and / or insertions, as appropriate)) via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988); incorporated herein by reference in its entirety).

[0167] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen-binding portions thereof can be recovered and purified by known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods, such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See generally, Scopes, Protein Purification (Springer-Verlag, New York, 1982). Substantially pure GPC3 antibodies or antigen-binding portions thereof of at least about 90%-95% homogeneity are advantageous, particularly for pharmaceutical applications, with 98%-99% or greater homogeneity being advantageous. The intact anti-GPC3 antibodies or antigen-binding portions thereof, once partially or homogeneously purified as desired, can then be used therapeutically, or in developing and performing assay procedures, immunofluorescence staining, and the like. See generally, Immunol.Meth. Vol. I and II (Lefkovits and Pernis, eds., Acad.Press, New York, 1979 and 1981).

[0168] Furthermore, as described herein, anti-GPC3 antibodies or antigen-binding portions thereof can be further optimized to reduce potential immunogenicity while maintaining functional activity for treatment in humans.

[0169] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 11 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 12, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 11 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 12, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12.

[0170] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 19, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 19, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19.

[0171] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 24, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 24, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0172] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 128 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 29, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 128 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 29, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 128 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 29.

[0173] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 34, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 34, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:34.

[0174] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 38, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 38, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 37 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 38.

[0175] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 44 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 45, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 44 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 45, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that can exhibit one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 44 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 45.

[0176] In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 51 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the optimized GPC3-binding antibody or antigen-binding portion thereof is derived from an anti-GPC3 antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 51 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy chain or light chain variable region are unmodified. In this regard, functional activity refers to an anti-GPC3 antibody or antigen-binding portion thereof that is capable of exhibiting one or more known functional activities associated with a GPC3-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 51 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52.

[0177] In any of these embodiments, the functional activity of the GPC3-binding antibody or antigen-binding portion thereof includes specific binding to GPC3. Further functional activities include anti-cancer activity. In addition, an anti-GPC3 antibody or antigen-binding portion thereof having functional activity has a specific binding activity, whether or not it is dose-dependent, when measured in a particular assay, e.g., a biological assay, that is greater than or equal to the reference antibody or antigen-binding portion thereof described herein (e.g., (i) a VH having an amino acid sequence as set forth in SEQ ID NO: 11 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 12; (ii) a VH having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 19; (iii) a VH having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 24; (iv) a VH having an amino acid sequence as set forth in SEQ ID NO: 128 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 29). (v) a VH having an amino acid sequence as set forth in SEQ ID NO: 1 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 34; (vi) a VH having an amino acid sequence as set forth in SEQ ID NO: 37 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 38; (vii) a VH having an amino acid sequence as set forth in SEQ ID NO: 44 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 45; or (viii) a VH having an amino acid sequence as set forth in SEQ ID NO: 51 and / or a VL having an amino acid sequence as set forth in SEQ ID NO: 52; or a variant thereof as described herein) of a GPC3-binding antibody or antigen-binding portion thereof. If a dose-dependence exists, it need not be identical to the dose-dependence of the reference antibody or antigen-binding portion thereof, but rather is substantially similar to or better than the dose-dependence in a given activity compared to the reference antibody or antigen-binding portion thereof as described herein (i.e., the candidate polypeptide will exhibit greater activity compared to the reference antibody).

[0178] II. Antibody Drug Conjugates In some embodiments, the anti-GPC3 (ARD103 variant) antibody is part of an anti-GPC3 antibody drug conjugate (or GPC3 conjugate). In some embodiments, the anti-GPC3 antibody is bound to at least one linker, and at least one cytotoxic agent is bound to each linker.

[0179] As used herein, a "cytotoxic agent" refers to a compound that exerts a cytotoxic or cytostatic effect on a cell, for example, by preventing cell growth or replication. A "small molecule" or "compound" is an organic compound having a molecular weight of less than 1500, or 100, or 900, or 750, or 600, or 500 daltons. A "small molecule drug" is a small molecule that has a therapeutic effect, such as treating a disease or disorder. In some embodiments, the small molecule is not a protein, polysaccharide, or nucleic acid.

[0180] In some embodiments, the cytotoxic agent is a microtubule disrupting agent (eg, a tubulin disrupting agent) or a DNA modifying agent.

[0181] In some embodiments, the GPC3 conjugate comprises a cytotoxic agent that is a tubulin disrupting agent. Several different categories of tubulin disrupting agents are known, including auristatins, tubulysins, colchicines, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterins, and other tubulin disrupting agents. Auristatins are derivatives of the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine or monomethylauristatin E) and MMAF (N-methylvaline-valine-dolaisoleucine-dolaproine-phenylalanine or monomethylauristatin F) and AFP (see PCT Publication Nos. WO2004 / 010957 and WO2007 / 008603). PCT Publication No. WO2015 / 057699 describes PEGylated auristatins containing MMAE. Additional dolastatin derivatives contemplated for use are disclosed in U.S. Patent No. 9,345,785, which is incorporated herein by reference.

[0182] Tubulysins include, but are not limited to, tubulysin D, tubulysin M, tubuphenylalanine and tubutyrosin. PCT Publication Nos. WO2017 / 096311 and WO2016 / 040684 describe tubulysin analogs, including tubulysin M.

[0183] Colchicine includes, but is not limited to, colchicine and CA-4.

[0184] Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VOS).

[0185] Taxanes include, but are not limited to, paclitaxel and docetaxel.

[0186] Cryptophycins include, but are not limited to, cryptophycin-1 and cryptophycin-52. Maytansinoids include, but are not limited to, maytansine, maytansinol, maytansine analogs in DM1, DM3 and DM4, and ansamatocin-2. Exemplary maytansinoid drug moieties include those with modified aromatic rings such as C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / - dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chloride), as well as those with modifications at other positions.

[0187] Maytansinoid drug moieties include C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (prepared from Nocardia sp.); C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces sp.); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared from Trewia sp.). nudiflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces sp.); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol). The cytotoxicity of TA.1-maytansonoid conjugates (Chari et al., Cancer Research 52:127-131 (1992)) binding to HER-2 was tested in vitro in the human breast cancer cell line SK-BR-3. The drug conjugates achieved a degree of cytotoxicity similar to that of the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule.

[0188] Hemiasterins include, but are not limited to, hemiasterin and HTL-286.

[0189] Other tubulin disrupting agents include taccalonolide A, taccalonolide B, taccalonolide AF, taccalonolide AJ, taccalonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0190] In some embodiments, the cytotoxic agent is a DNA modifying agent. In some embodiments, the DNA modifying agent is an alkylating agent or a topoisomerase inhibitor. In some embodiments, the DNA modifying agent is a duocarmycin or an analog thereof, a calicheamicin, or a pyrrolobenzodiazepine.

[0191] In some embodiments, the cytotoxic agent may be a topoisomerase inhibitor, such as camptothecin or a camptothecin analog, or an anthracycline. Camptothecin or its analogs include irinotecan (also called CPT-11), topotecan, 10-hydroxy-CPT, SN-38, exatecan and exatecan analog DXd (see US Patent Application Publication No. 2015 / 0297748). Examples of anthracyclines include doxorubicin, epirubicin, nembicin; PNU-159682 and its derivatives (see US Patent No. 10,960,083; Quintieri et al. (2005) Clin.Cancer Res. 11:1608-1617; Stefan et al. (2017) Mol.Cancer Ther. 16:879-892).

[0192] In some embodiments, the cytotoxic agent is a duocarmycin, including the synthetic analogs, KW-2189 and CBI-TMI.

[0193] The GPC3 conjugates contemplated for use in the methods herein comprise at least one linker, with each linker having at least one cytotoxic agent attached thereto.Typically, the conjugate comprises a linker between the anti-GPC3 antibody or its antigen-binding fragment and the cytotoxic agent.The linker may be a protease-cleavable linker (see, for example, PCT Publication No. WO2004 / 010957), an acid-cleavable linker, a disulfide linker, a self-stabilizing linker (see, for example, PCT Publication No. WO2018 / 031690 and WO2015 / 095755), a non-cleavable linker (see, for example, PCT Publication No. WO2007 / 008603), and / or a hydrophilic linker (see, for example, PCT Publication No. WO2015 / 123679). In various embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the cytotoxic agent from the antibody in the intracellular environment.

[0194] For example, in some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to a lysosomal or endosomal protease. Typically, the peptidyl linker is at least one amino acid long or at least two amino acids long. Cleavage agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most typical peptidyl linker is a peptidyl linker that is cleavable by an enzyme present in the target antigen-expressing cell. For example, a peptidyl linker cleavable by cathepsin B, a thiol-dependent protease highly expressed in cancerous tissues, can be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 95) linker). Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a val-cit linker) or a Gly-Gly-Phe-Gly (SEQ ID NO: 96) linker (see, for example, U.S. Patent Application Publication No. 2015 / 0297748). One advantage of using intracellular proteolytic release of a cytotoxic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high. See also U.S. Patent No. 9,345,785.

[0195] As used herein, the terms "cleaved intracellularly" and "intracellular cleavage" refer to a metabolic process or reaction within a cell on an antibody drug conjugate whereby the covalent bond, e.g., linker, between the cytotoxic agent and the antibody is cleaved to yield free cytotoxic agent or other metabolic product of the conjugate dissociated from the antibody within the cell. Thus, the cleaved portion of the conjugate is an intracellular metabolic product.

[0196] In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to a therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0197] In various embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known, including those that can be formed using, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene) (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (ed. C.W. Vogel, Oxford U. Press, 1987. U.S. Pat. No. 4,880,935).

[0198] In various embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). In some embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See U.S. Patent Application Publication No. 2005 / 0238649).

[0199] In various embodiments, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a linker means that about 20% or less, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of an antibody drug conjugate (ADC) or ADC derivative are cleaved when the ADC or ADC derivative is present in an extracellular environment (e.g., in plasma). Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) an ADC or ADC derivative ("ADC sample") and (b) an equal molar amount of unconjugated antibody or therapeutic agent "control sample" with plasma independently for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then measuring the amount of unconjugated antibody or therapeutic agent present in the ADC sample, for example, by high performance liquid chromatography, and comparing it to the amount present in the control sample.

[0200] In various embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to a cytotoxic agent (i.e., in the context of the linker-therapeutic agent portion of an ADC or ADC derivative described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both a cytotoxic agent and an anti-GPC3 antibody or derivative thereof (i.e., in the context of an ADC or ADC derivative as described herein).

[0201] Various linkers that can be used with the compositions and methods of the present invention are described in PCT Publication No. WO2004010957. In various embodiments, the protease cleavable linker comprises a thiol-reactive spacer and a dipeptide. In some embodiments, the protease cleavable linker is composed of a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, and a p-aminobenzyloxycarbonyl spacer.

[0202] In various embodiments, the acid-cleavable linker is a hydrazine linker or a quaternary ammonium linker (see PCT Publication Nos. WO2017 / 096311 and WO2016 / 040684).

[0203] Self-stabilizing linkers containing maleimide groups are described in US Pat. No. 9,504,756.

[0204] In various embodiments, the tubulin disrupting agent, such as auristatin, is conjugated to the linker by its C-terminal carboxyl group forming an amide bond with the linker unit (LU), as described in U.S. Patent No. 9,463,252, which is incorporated herein by reference. In various embodiments, the linker unit comprises at least one amino acid. N,N-dialkyl auristatin binder-drug conjugates (ADCs) are disclosed in U.S. Patent No. 8,992,932.

[0205] In various embodiments, the linker also includes a stretcher unit and / or an amino acid unit. Exemplary stretcher units and amino acid units are described in U.S. Patent Nos. 9,345,785 and 9,078,931, each of which is incorporated herein by reference.

[0206] In various embodiments, the present disclosure provides an antibody-drug conjugate comprising an anti-GPC3 antibody covalently linked to MMAE via a mc-val-cit-PAB linker. The GPC3 conjugate is delivered to a subject as a pharmaceutical composition.

[0207] In some embodiments, the GPC3 conjugate has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein mAb is an anti-GPC3 antibody, S is a sulfur atom of the antibody, A- is a stretcher unit, and p is about 3 to about 5, or about 3 to about 8.

[0208] Drug loading is represented by p, the average number of drug molecules (cytotoxic agents) per antibody in the pharmaceutical composition. For example, if p is about 4, then the average drug loading considering all of the antibodies present in the pharmaceutical composition is about 4. In some embodiments, P ranges from about 3 to about 5, more preferably from about 3.6 to about 4.4, and even more preferably from about 3.8 to about 4.2. P may be about 3, about 4, or about 5. In some embodiments, P ranges from about 6 to about 8, more preferably from about 7.5 to about 8.4. P may be about 6, about 7, or about 8. The average number of drugs per antibody in the preparation of the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of antibody-drug conjugates with respect to p can also be determined. In some examples, separation, purification, and characterization of homogeneous antibody-drug conjugates with one value of p and antibody-drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0209] The stretcher unit (A) can link the antibody unit to the amino acid unit (e.g., valine-citrulline peptide) via a sulfhydryl group of the antibody. The sulfhydryl group can be generated, for example, by reduction of the interchain disulfide bond of the anti-GPC3 antibody. For example, the stretcher unit can be linked to the antibody via a sulfur atom resulting from reduction of the interchain disulfide bond of the antibody. In some embodiments, the stretcher unit is linked to the antibody only via a sulfur atom resulting from reduction of the interchain disulfide bond of the antibody. In some embodiments, the sulfhydryl group can be generated by reaction of the amino group of the lysine moiety of the anti-GPC3 antibody with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagent. In certain embodiments, the anti-GPC3 antibody is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant anti-GPC3 antibody is engineered to carry additional sulfhydryl groups, for example additional cysteines.

[0210] The synthesis and structure of MMAE are described in U.S. Patent No. 6,884,869, which is incorporated herein by reference in its entirety for all purposes. The synthesis and structure of exemplary Stretcher units and methods for making antibody-drug conjugates are described, for example, in U.S. Patent Application Publication Nos. 2006 / 0074008 and 2009 / 0010945, each of which is incorporated herein by reference in its entirety.

[0211] Representative stretcher units are described within the brackets of formulas Illa and lllb of U.S. Pat. No. 9,211,319, incorporated herein by reference.

[0212] In various embodiments, the antibody drug conjugate comprises monomethyl auristatin E and a protease cleavable linker. The protease cleavable linker is believed to comprise a thiol-reactive spacer and a dipeptide. In various embodiments, the protease cleavable linker is comprised of a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, and a p-amino-benzyloxycarbonyl or PAB spacer.

[0213] The abbreviation "MMAE" refers to monomethylauristatin E.

[0214] The abbreviations "vc" and "val-cit" refer to the dipeptide valine-citrulline.

[0215] The abbreviation "PAB" stands for self-immolating spacer: [ka]

[0216] The abbreviation "MC" refers to the stretcher maleimidocaproyl; [ka]

[0217] In other exemplary embodiments, the conjugate has the general formula: Ab-[L3]-[L2]-[L1] m -AA n -cytotoxic agents, where Ab is an anti-GPC3 antibody; the cytotoxic agent may be a tubulin disrupting agent or a topoisomerase inhibitor; L3 is a component of a linker that includes an antibody coupling moiety and one or more acetylene (or azide) groups; L2 includes a defined PEG (polyethylene glycol) azide (or acetylene) at one end that is complementary to the acetylene (or azide) moiety of L3, and a reactive group such as a carboxylic acid or hydroxyl group at the other end; L1 includes a foldable unit (e.g., a self-immolative group), or a peptidase-cleavable moiety, or an acid-cleavable moiety, optionally linked to the foldable unit; AA is an amino acid; m is an integer having a value of 0 or 1, and n is an integer having a value of 0, 1, 2, 3, or 4. Such linkers can be assembled by click chemistry. (See, for example, U.S. Patent Nos. 7,591,944 and 7,999,083.)

[0218] In some embodiments, the cytotoxic agent is camptothecin or a camptothecin (CPT) analog, such as irinotecan (also called CPT-11), topotecan, 10-hydroxy-CPT, exatecan, DXd, and SN-38. Representative structures are shown below. [ka]

[0219] Conjugates of the formula Ab-[L3]-[L2]-[L1] m -AA n- With reference to the cytotoxic agent, in some embodiments, m is 0. In such embodiments, an ester moiety is first formed between the carboxylic acid of an amino acid (AA), such as glycine, alanine, or sarcosine, or the carboxylic acid of a peptide, such as glycylglycine, and a hydroxyl group of the cytotoxic agent. In this example, the N-terminus of the amino acid or polypeptide can be protected as a Boc or Fmoc or monomethoxytrityl (MMT) derivative, which is deprotected after formation of the ester bond with the hydroxyl group of the cytotoxic agent. Since "MMT" is removable by mild acid treatment, such as dichloroacetic acid, which does not cleave the BOC group, selective removal of the amine protecting group can be achieved in the presence of a BOC protecting group at the hydroxyl position of the cytotoxic agent containing an additional hydroxyl group, using monomethoxytrityl (MMT) as a protecting group for the amino group of the amino acid or polypeptide involved in the ester formation. After the amino group of the amino acid or polypeptide that forms the ester bond with the hydroxyl of the cytotoxic agent is unmasked, the amino group is reacted with the activated form of the COOH group on the PEG moiety of L2 under standard amide formation conditions. In a preferred embodiment, L3 comprises a thiol reactive group that binds to the thiol group of the antibody. The thiol reactive group is optionally maleimide or vinylsulfone, or bromoacetamide, or iodoacetamide that binds to the thiol group of the antibody. In some embodiments, the reagent with the thiol reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, and the thiol reactive group is a maleimide group.

[0220] In another embodiment, m is 0 and AA comprises a peptide moiety, preferably a di-, tri- or tetrapeptide, that is cleavable by an intracellular peptidase, such as cathepsin B. Examples of cathepsin B cleavable peptides are Phe-Lys, Val-Cit (Dubowchick, 2002), Ala-Leu, Leu-Ala-Leu and Ala-Leu-Ala-Leu (SEQ ID NO: 97) (Trouet et al., 1982).

[0221] In a preferred embodiment, L1 is composed of an intracellularly cleavable peptide, such as a cathepsin B cleavable peptide, bound at the C-terminus of the peptide to a foldable unit p-aminobenzyl alcohol (or p-amino-benzyloxycarbonyl), the benzyl alcohol moiety of which is directly bound to the hydroxyl group of the cytotoxic agent in chloroformate form. In this embodiment, n is 0. Alternatively, if "n" is not zero, the benzyl alcohol moiety of the p-amidobenzyl alcohol (or p-amino-benzyloxycarbonyl) moiety is bound to the N-terminus of the amino acid or peptide to which it is linked at the hydroxyl group of the cytotoxic agent via the activated form of p-amidobenzyl alcohol, i.e., PABOCOPNP, where PNP is p-nitrophenyl. In a preferred embodiment, the linker comprises a thiol reactive group that binds to the thiol group of the antibody. The thiol reactive group is optionally a maleimide or vinyl sulfone, or a bromoacetamide, or an iodoacetamide that binds to the thiol group of the antibody. In a preferred embodiment, the component having a thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, where the thiol-reactive group is a maleimide group.

[0222] In a preferred embodiment where the cytotoxic agent is a camptothecin or analog or derivative thereof having a 20-hydroxyl, L1 is composed of an intracellularly cleavable peptide such as a cathepsin B cleavable peptide linked at the C-terminus of the peptide to a foldable linker p-aminobenzyl alcohol (or p-amino-benzyloxycarbonyl), the benzyl alcohol moiety of which is directly linked to CPT-20-O-chloroformate. In this embodiment, n is 0. Alternatively, if "n" is not zero, the benzyl alcohol moiety of the p-amidobenzyl alcohol moiety is linked to the N-terminus of the amino acid or peptide linked at the 20-position of CPT via the activated form of p-amidobenzyl alcohol, i.e., PABOCOPNP, where PNP is p-nitrophenyl. In a preferred embodiment, the linker comprises a thiol reactive group which is linked to a thiol group of an antibody. The thiol reactive group is optionally a maleimide or vinyl sulfone, or a bromoacetamide, or an iodoacetamide, which is linked to a thiol group of an antibody. In a preferred embodiment, the component having a thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, where the thiol-reactive group is a maleimide group.

[0223] In another embodiment, the L2 component of the conjugate contains a polyethylene glycol (PEG) spacer that may be up to MW 5000 in size, in a preferred embodiment, the PEG is a defined PEG having (1-12 or 1-30) repeating monomer units. In a further preferred embodiment, the PEG is a defined PEG having 1-12 repeating monomer units. The introduction of PEG may include the use of commercially available heterobifunctional PEG derivatives. In the context of this disclosure, heterobifunctional PEGs contain azide or acetylene groups. An example of a heterobifunctional defined PEG containing 8 repeating monomer units where "NHS" is succinimidyl is shown in the formula below. [ka]

[0224] In a preferred embodiment, L3 has a plurality of acetylene (or azide) groups, in the range of 2 to 40, preferably 2 to 20, more preferably 2 to 5, and a single antibody binding moiety.

[0225] A representative conjugate is shown below in which the cytotoxic agent is SN-38 (a CPT analog) and was prepared using a maleimide-containing SN-38-linker derivative, with the attachment to the antibody (designated MAb) represented as the succinimide, where m=0 and the 20-O-AA ester attached to SN-38 is a glycinate; an azide-acetylene coupling bond between L2 and L3 results in a triazole moiety as shown. [ka]

[0226] In another representative conjugate, prepared with a maleimide-containing SN-38-linker derivative, the linkage to the antibody (MAb) is depicted as a succinimide, as shown below: where in general formula 2, n=0; "L1" contains a cathepsin B cleavable dipeptide linked to a foldable p-aminobenzyl alcohol moiety, the former being linked to SN-38 as a carbonate bond at position 20; and an azide-acetylene coupling linking the "L2" and "L3" moieties results in a triazole moiety as shown. [ka]

[0227] Another representative SN-38 conjugate, Mab-CL2-SN-38, prepared with a maleimide-containing SN-38 linker derivative and in which the linkage to the antibody is represented as a succinimide, is shown below, where the 20-O-AA ester attached to SN-38 is a p-aminobenzyl alcohol moiety and a glycinate attached to the L1 moiety via a cathepsin-B cleavable dipeptide; the latter is attached to "L2" via an amide bond, while the "L2" and "L3" moieties are linked via azide-acetylene "click chemistry." They are bonded together. [ka]

[0228] Another example of a preferred embodiment is shown below, where "L1" comprises a single amino acid attached to a foldable p-aminobenzyl alcohol moiety, where the p-aminobenzyl alcohol is substituted or unsubstituted (R), where m=1 and n=0 in the general conjugate formula, and where the cytotoxic agent is exemplified by SN-38. The structure is shown below (referred to as MAb-CLX-SN-38). The single amino acid of AA can be selected from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The substituent R of the 4-aminobenzyl alcohol moiety is hydrogen or an alkyl group selected from C1-C10 alkyl groups. [ka]

[0229] Shown below is an embodiment of MAb-CLX-SN-38 (above) in which the single amino acid AA is L-lysine, R=H, and the cytotoxic agent is exemplified by SN-38 (referred to as MAb-CL2A-SN-38). [ka]

[0230] In other embodiments, the cytotoxic agent is attached to a linker comprising a stretcher unit (Z) attached to an amino acid unit (AA) attached to a spacer unit (Y), where the stretcher unit is attached to the antibody (Ab or MAb) and the spacer unit is attached to an amino group of the cytotoxic agent. Such linkers have the formula: Ab-Z-AA-Y-cytotoxic agent, In the formula, Z is -(succinimid-3-yl-N)--(CH2) n 2 -C(=O)--, -CH2--C(=O)--NH--(CH2)n 3 -C(=O)--, -C(=O)-cyc.Hex(1,4)-CH2--(N-ly-3-diminiccuS)- or -C(=O)--(CH2)n 4 -C(=O)--, where n 2 represents an integer from 2 to 8, and n 3 represents an integer from 1 to 8, and n 4 represents an integer of 1 to 8; cyc.Hex(1,4) represents a 1,4-cyclohexylene group; (N-ly-3-diminiccuS)- has a structure represented by the following formula: [ka]

[0231] AA is a peptide of 2 to 7 amino acids. The spacer unit Y is -NH-(CH2) b -(C=O)- or -NH-CH2-O-CH2-(C=O)-, where b is an integer of 1 to 5.

[0232] In some embodiments, the cytotoxic agent is exatecan. In some embodiments, the amino acid unit (AA) is -Gly-Gly-Phe-Gly- (SEQ ID NO: 96). In some embodiments, the spacer unit Y is -NH-CH2-O-CH2-(C=O)-.

[0233] In some embodiments, the linker-cytotoxic agent has the structure: [ka] Here, the cytotoxic agent released is DXd (see US Pat. No. 9,808,537).

[0234] Conjugation of a cytotoxic agent-linker to an antibody or antibody-binding moiety Techniques for attaching a cytotoxic agent to an antibody or its antigen-binding portion via a linker are well known in the art. See, for example, Alley et al., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3):154-169. In some embodiments, the linker is attached to the cytotoxic agent first, and then the linker-cytotoxic agent is attached to the antibody or its antigen-binding portion. In some embodiments, the linker is attached to the antibody or its antigen-binding portion first, and then the cytotoxic agent is attached to the linker. In the following discussion, the term linker-cytotoxic agent is used to illustrate the attachment of a linker or linker-cytotoxic agent to an antibody or its antigen-binding portion, and one of skill in the art will understand that the attachment method selected may be selected according to the linker and cytotoxic agent. In some embodiments, the cytotoxic agent is attached to the antibody or its antigen-binding portion via a linker in a manner that reduces its activity until it is released from the conjugate (e.g., by hydrolysis, by proteolysis, or by a cleavage agent).

[0235] In general, the conjugates can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including (1) reacting a nucleophilic group of an antibody or its antigen-binding portion with a bivalent linker reagent to form an antibody-linker intermediate via a covalent bond, followed by reaction with a cytotoxic agent, and (2) reacting a nucleophilic group of a cytotoxic agent with a bivalent linker reagent to form a linker-cytotoxic agent via a covalent bond, followed by reaction with a nucleophilic group of an antibody or its antigen-binding portion. An exemplary method for preparing conjugates via the latter route is described in U.S. Pat. No. 7,498,298, which is expressly incorporated herein by reference.

[0236]

[0308] Nucleophilic groups on antibodies include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups on which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl and benzyl halides, such as haloacetamides, and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with reducing agents, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), so that the antibody is fully or partially reduced. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, for example, by reacting the lysine residues with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Reactive thiol groups can also be introduced into antibodies by introducing one, two, three, four or more cysteine ​​residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine ​​amino acid residues).

[0237] The conjugates of the present disclosure can also be produced by the reaction of electrophilic groups on an antibody, such as aldehyde or ketone carbonyl groups, with nucleophilic groups on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugar of a glycosylated antibody can be oxidized, for example with a periodate oxidation reagent, to form an aldehyde or ketone group that can react with an amine group on a linker reagent or drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, for example, with a borohydride reagent, to form a stable amine bond. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can result in carbonyl (aldehyde and ketone) groups in the antibody or its antigen-binding portion that can react with appropriate groups on a drug (see, e.g., Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to produce an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Patent No. 5,362,852). Such aldehydes can be reacted with cytotoxic agents or linkers.

[0238] Exemplary nucleophilic groups on a cytotoxic agent include, but are not limited to, (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups.

[0239] Non-limiting exemplary cross-linking reagents that can be used to prepare conjugates are described herein or known to those skilled in the art. Methods of using such cross-linking reagents to link two moieties, including proteinaceous and chemical moieties, are known in the art. In some embodiments, a fusion protein comprising an antibody and a cytotoxic agent can be produced, for example, by recombinant technology or peptide synthesis. A recombinant DNA molecule can comprise a region that codes for an antibody and a region that codes for the cytotoxic moiety of the conjugate, adjacent to each other or separated by a region that codes for a linker peptide that does not destroy the desired properties of the conjugate.

[0240] In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, and the antibody-receptor conjugate is administered to the patient, followed by removing unbound conjugate from the circulation using a detergent, and then administering a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or radioactive nucleotide).

[0241] In some embodiments, the linker-cytotoxic agent is attached to an interchain cysteine ​​residue of the antibody or antigen-binding fragment thereof. See, for example, PCT Publication Nos. WO2004 / 010957 and WO2005 / 081711. In such embodiments, the linker typically comprises a maleimide group for attachment to the cysteine ​​residue of the interchain disulfide. In some embodiments, the linker or linker-cytotoxic agent is attached to a cysteine ​​residue of the antibody or antigen-binding portion thereof, as described in U.S. Pat. Nos. 7,585,491 or 8,080,250. The drug loading of the resulting conjugate is typically in the range of 1-8.

[0242] In some embodiments, the linker or linker-cytotoxic agent is attached to a lysine or cysteine ​​residue of the antibody or antigen-binding portion thereof, as described in PCT Publication Nos. WO2005 / 037992 or WO2010 / 141566. The drug loading of the resulting conjugate is typically in the range of 1-8.

[0243] In some embodiments, engineered cysteine ​​residues, polyhistidine sequences, glycoengineered tags or transglutaminase recognition sequences can be used for site-specific attachment of a linker or linker-cytotoxic agent to an antibody or antigen-binding portion thereof.

[0244] In some embodiments, the linker-cytotoxic agent is attached to an engineered cysteine ​​residue at an Fc region residue other than the interchain disulfide. In some embodiments, the linker-cytotoxic agent is attached to an engineered cysteine ​​introduced into an IgG (typically an IgG1) at heavy chain positions 118, 221, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324 and / or at position 106, 108, 142 (light chain), 149 (light chain) and / or at position V205. An exemplary substitution for site-specific conjugation using an engineered cysteine ​​is S239C (see, e.g., US Patent Application Publication No. 2010 / 0158909; numbering of the Fc region is according to the EU index).

[0245] In some embodiments, the linker or linker-cytotoxic agent is attached to one or more introduced cysteine ​​residues of the antibody or antigen-binding portion thereof, as described in PCT Publication Nos. WO2006 / 034488, WO2011 / 156328 and / or WO2016040856.

[0246] In some embodiments, the exemplary substitution for site-specific conjugation using bacterial transglutaminase is N297S or N297Q in the Fc region. In some embodiments, the linker or linker-cytotoxic agent is attached to the glycan or modified glycan of the antibody or antigen-binding portion or glycoengineered antibody or antigen-binding portion thereof. For example, see PCT Publication Nos. WO2017 / 147542, WO2020 / 123425, WO2014 / 072482, WO2014 / 065661, WO2015 / 057066 and WO2016 / 022027.

[0247] III. Pharmaceutical Preparations Another aspect of the anti-GPC3 antibody and its antigen-binding portion or other binding agent relates to a composition comprising an active ingredient (i.e., an anti-GPC3 antibody or its antigen-binding portion or other binding agent described herein or a conjugate thereof, or a nucleic acid encoding an antibody or its antigen-binding portion or other binding agent described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, diluent, or excipient approved for use in the pharmaceutical industry. The phrase "pharmaceutical acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0248] The preparation of pharmacological compositions containing dissolved or dispersed active ingredients is well understood in the art and need not be limited based on a particular formulation. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for rehydration or suspension in liquid prior to use can also be prepared. The preparations may also be emulsified or presented as liposomal compositions. The anti-GPC3 antibody or its antigen-binding portion or other binding agent or conjugate thereof can be mixed with a pharmaceutically acceptable excipient compatible with the active ingredient in an amount suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the pharmaceutical composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., anti-GPC3 antibody or its antigen-binding portion). The pharmaceutical compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as, for example, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically acceptable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing active ingredient (e.g., anti-GPC3 antibody and / or its antigen-binding portion or conjugate) and water, and may contain buffers such as sodium phosphate, saline or both at physiological pH values, for example, phosphate buffered saline. In addition, aqueous carriers may contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.Liquid compositions can also contain liquid phases in addition to and other than water.Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.The amount of active agent that will be effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0249] The pharmaceutical compositions described herein can be formulated for oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intrasternal and intratumoral injection or infusion techniques.

[0250] In some embodiments, the pharmaceutical composition of the present disclosure is formulated in the form of single dosage unit or multiple dosage units.The method of preparing such dosage form is known or clear to those skilled in the art, for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000).

[0251] In some embodiments, the pharmaceutical composition comprising the anti-GPC3 antibody or antigen-binding portion thereof or conjugate thereof described herein, or a nucleic acid encoding the anti-GPC3 antibody or antigen-binding portion thereof described herein, may be lyophilized.

[0252] In some embodiments, a syringe containing a therapeutically effective amount of an anti-GPC3 antibody or an antigen-binding portion thereof or a conjugate thereof, or a pharmaceutical composition described herein is provided. IV. Therapeutic Uses of Anti-GPC3 Antibodies, Antigen-Binding Portions Thereof, Binding Agents, and Conjugates In some aspects, the anti-GPC3 antibodies or antigen-binding portions thereof, binding agents and conjugates described herein can be used in a method comprising administering the anti-GPC3 antibodies or antigen-binding portions thereof or other binding agents or conjugates described herein to a subject in need of administration. In some embodiments, the anti-GPC3 antibodies or antigen-binding portions thereof include: (i) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 12; (ii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 128 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 29. (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52.

[0253] In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof comprises: (i) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 12; (ii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 128 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 34. (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52, wherein the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.In some embodiments, the anti-GPC3 antibody or antigen-binding portion thereof comprises: (i) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 12; (ii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 128 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain variable (VL) region having an amino acid sequence as set forth in SEQ ID NO: 34. (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52, wherein the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. The GPC3 conjugate comprises an antibody or antigen-binding portion of any of these embodiments.

[0254] In some embodiments, the subject is in need of treatment for cancer and / or malignant tumors. In some embodiments, the subject is in need of treatment for GPC3+ cancer or GPC3+ malignant tumors, such as lung cancer, such as hepatocellular carcinoma, small cell lung cancer, lung squamous cell carcinoma, and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, vulvar cancer, renal cell carcinoma, breast cancer (e.g., triple negative breast cancer), melanoma, germ cell cancer (e.g., testicular), gastric cancer, sarcoma, and bladder cancer. In some embodiments, the method is for treating a subject with a GPC3+ cancer or malignant tumor. In some embodiments, the method is for treating hepatocellular carcinoma in a subject. In some embodiments, the method is for treating lung cancer, such as small cell lung cancer, small cell lung cancer, and large cell lung cancer in a subject. In some embodiments, the method is for treating colorectal cancer in a subject. In some embodiments, the method is for treating esophageal cancer in a subject. In some embodiments, the method is for treating cervical cancer in a subject. In some embodiments, the method is for treating head and neck cancer in a subject. In some embodiments, the method is for treating ovarian cancer in a subject. In some embodiments, the method is for treating vulvar cancer in a subject. In some embodiments, the method is for treating renal cell carcinoma in a subject. In some embodiments, the method is for treating breast cancer in a subject. In some embodiments, the method is for treating triple negative breast cancer in a subject. In some embodiments, the method is for treating melanoma in a subject. In some embodiments, the method is for treating germ cell cancer in a subject. In some embodiments, the method is for treating sarcoma in a subject. In some embodiments, the method is for treating gastric cancer in a subject. In some embodiments, the method is for treating bladder cancer in a subject.

[0255] The methods described herein include administering a therapeutically effective amount of an anti-GPC3 antibody or its antigen-binding portion or other binding agent or conjugate to a subject with a GPC3+ cancer or malignant tumor.As used herein, the phrase "therapeutically effective amount", "effective amount" or "effective dose" refers to the amount of an anti-GPC3 antibody or its antigen-binding portion or other binding agent or conjugate described herein that provides a therapeutic benefit in the treatment, management or prevention of recurrence of a cancer or malignant tumor, for example, an amount that provides a statistically significant reduction in at least one symptom, sign or marker of a tumor or malignant tumor.The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art.In general, a therapeutically effective amount may vary depending on the subject's medical history, age, symptoms, sex, and the severity and type of the subject's medical condition, as well as the administration of other pharmacoactive agents.

[0256] The terms "cancer" and "malignancy" refer to the uncontrolled proliferation of cells that interferes with the normal function of the body's organs and systems. Cancer or malignant tumors may be primary or metastatic, i.e., have become invasive, seeding tumor growth in tissues distant from the original tumor site. "Tumor" refers to the uncontrolled proliferation of cells that interferes with the normal function of the body's organs and systems. A subject with cancer is one that has objectively measurable cancer cells present in the subject's body. This definition includes benign and malignant tumors, as well as potentially dormant tumors and micrometastases. Cancers that migrate from their original location and seed other vital organs may ultimately result in the death of the subject through the compromised function of the affected organ. Hematological malignancies (hematopoietic cancers), such as leukemia and lymphoma, can overwhelm the subject's normal hematopoietic compartment, thereby resulting in hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) and ultimately causing death.

[0257] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer (e.g., triple-negative breast cancer), peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, chondrosarcoma, colon and rectal cancer (colorectal cancer), connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer (gastrointestinal cancer and stomach cancer). cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer or renal cancer (e.g., clear cell carcinoma), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, cancer of the respiratory system, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, uterine cancer, urinary system cancer, vulvar cancer, and other cancers and sarcomas, as well as B-cell lymphoma. Examples of lymphomas include, but are not limited to, lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus syndrome, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0258] In some embodiments, the cancer is selected from solid tumors, including, but not limited to, hepatocellular carcinoma, lung cancer, such as small cell lung cancer, squamous cell lung cancer, and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, renal cell carcinoma, breast cancer (e.g., triple negative breast cancer), melanoma, germ cell cancer (e.g., testicular), vulvar cancer, stomach cancer, sarcoma, and bladder cancer.

[0259] In some embodiments, cancer or malignant tumor is GPC3 positive (GPC3+).As used herein, the term "GPC3 positive" or "GPC3+" is used to describe cancer cells, clusters of cancer cells, tumor masses, or metastatic cells that express GPC3 on the cell surface (membrane-bound GPC3).Some non-limiting examples of GPC3 positive cancer include hepatocellular carcinoma, lung cancer such as small cell lung cancer, squamous cell lung cancer, and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, renal cell carcinoma, breast cancer (e.g., triple-negative breast cancer), melanoma, germ cell cancer (e.g., testicular), vulvar cancer, gastric cancer, sarcoma, and bladder cancer.

[0260] The methods herein are contemplated to reduce tumor size or tumor burden in a subject and / or reduce metastasis in a subject. In various embodiments, the tumor size in the subject is reduced by about 25-50%, about 40-70%, or about 50-90% or more. In various embodiments, the methods reduce tumor size by 10%, 20%, 30% or more. In various embodiments, the methods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0261] As used herein, "subject" refers to a human or an animal. Usually, an animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient", "individual" and "subject" are used interchangeably herein.

[0262] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects to represent animal models, such as, for example, various cancers. Furthermore, the methods described herein can be used to treat livestock and / or pets. The subject may be male or female. In certain embodiments, the subject is a human.

[0263] The subject may be a person who has been previously diagnosed with GPC3+ cancer or has GPC3+ cancer and is in need of treatment, but does not need to have already been treated for GPC3+ cancer. Alternatively, the subject may also be a subject who has not been previously diagnosed with GPC3+ cancer in need of treatment. The subject may be a subject who exhibits one or more risk factors for a symptom or one or more complications associated with GPC3+ cancer, or a subject who exhibits no risk factors. A subject "in need" of treatment for GPC3+ cancer may be a subject who has the symptom or has been diagnosed with the symptom. In other embodiments, a subject "at risk of developing" a condition refers to a subject who has been diagnosed as being at risk of developing a condition (e.g., GPC3+ cancer).

[0264] As used herein, the terms "treat", "treatment", "treating" or "amelioration", when used in reference to a disease, disorder or medical condition, refer to therapeutic treatment of a condition with the goal of reversing, alleviating, improving, inhibiting, slowing down or halting the progression or severity of the symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of the condition. A treatment is generally "effective" if one or more symptoms or clinical markers are alleviated. Alternatively, a treatment is "effective" if the progression of the condition is alleviated or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least the slowing or worsening of the progression of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in GPC3+ cancer cells in a subject, alleviation of one or more symptoms, a reduction in the extent of the defect, a stabilized (i.e., not worsening) state of the cancer or malignant tumor, prevention or delay of tumor growth and / or metastasis, and an extension of lifespan compared to the lifespan expected in the absence of treatment. As used herein, the term "administering" refers to providing a subject with a nucleic acid encoding a GPC3-binding antibody or its antigen-binding portion or other binding agent or conjugate described herein, or an anti-GPC3 antibody or its antigen-binding portion or other binding agent described herein, by a method or route that results in the binding of the GPC3-binding antibody or its antigen-binding portion or other binding agent or conjugate to GPC3+ cancer cells or malignant cells.Similarly, a pharmaceutical composition comprising a GPC3-binding antibody or its antigen-binding portion or other binding agent or conjugate described herein, or a nucleic acid disclosed herein that encodes an anti-GPC3 antibody or its antigen-binding portion or other binding agent described herein, can be administered by any suitable route that results in an effective treatment for the subject.

[0265] The dosage range of the anti-GPC3 antibody or antigen-binding portion thereof or binding agent or conjugate depends on potency and includes an amount sufficient to produce the desired effect, e.g., tumor growth delay or tumor size reduction. The dosage should not be so large as to cause unacceptable adverse side effects. In general, the dosage will vary according to the age, condition and sex of the subject and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complications. In some embodiments, the dosage is in the range of 0.1 mg / kg body weight to 10 mg / kg body weight. In some embodiments, the dosage is in the range of 0.5 mg / kg body weight to 15 mg / kg body weight. In some embodiments, the dosage range is 0.5 mg / kg body weight to 5 mg / kg body weight. Alternatively, the dosage range can be titrated to maintain a serum level of 1 μg / mL to 1000 μg / mL. When administered systemically, a subject can be administered a therapeutic amount, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 12 mg / kg or more.

[0266] The administration of the above dose can be repeated.In a preferred embodiment, the dose listed above is administered every week, every other week, every three weeks or every month for several weeks or months.The duration of treatment depends on the clinical progress of the subject and its responsiveness to treatment.

[0267] In some embodiments, the dose may be from about 0.1 mg / kg to about 100 mg / kg. In some embodiments, the dose may be from about 0.1 mg / kg to about 25 mg / kg. In some embodiments, the dose may be from about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the dose may be from about 0.1 mg / kg to about 15 mg / kg. In some embodiments, the dose may be from about 0.1 mg / kg to about 12 mg / kg. In some embodiments, the dose may be from about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose may be from about 1 mg / kg to about 25 mg / kg. In some embodiments, the dose may be from about 1 mg / kg to about 20 mg / kg. In some embodiments, the dose may be from about 1 mg / kg to about 15 mg / kg. In some embodiments, the dose may be from about 1 mg / kg to about 12 mg / kg. In some embodiments, the dose may be about 2 mg / kg. In some embodiments, the dose may be about 4 mg / kg. In some embodiments, the dose may be about 5 mg / kg. In some embodiments, the dose may be about 6 mg / kg. In some embodiments, the dose may be about 8 mg / kg. In some embodiments, the dose may be about 10 mg / kg. In some embodiments, the dose may be about 10 mg / kg. In some embodiments, the dose may be about 12 mg / kg. In some embodiments, the dose may be about 100 mg / m 2 ~about 700mg / m 2 In some embodiments, the dose is about 250 mg / m 2 In some embodiments, the dose is about 375 mg / m 2 In some embodiments, the dose is about 400 mg / m 2 In some embodiments, the dose is about 500 mg / m 2 may be also possible.

[0268] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes.

[0269] In some embodiments, the doses can be administered weekly. In some embodiments, the doses can be administered every other week. In some embodiments, the doses can be administered about every two weeks. In some embodiments, the doses can be administered about every three weeks. In some embodiments, the doses can be administered every three weeks. In some embodiments, the doses can be administered every four weeks.

[0270] In some embodiments, about 2 to about 10 total doses are administered to the subject. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, a total of 9 doses are administered. In some embodiments, a total of 10 doses are administered. In some embodiments, more than 10 total doses are administered.

[0271] The pharmaceutical composition containing anti-GPC3 antibody or its antigen-binding portion or other GPC3 binding agent or GPC3 conjugate can be administered in unit dose.The term "unit dose" when used in relation to pharmaceutical composition refers to a physically separate unit suitable as a subject unitary dosage, each unit containing a predetermined amount of active material (e.g., anti-GPC3 antibody or its antigen-binding portion or conjugate) calculated to produce the desired therapeutic effect in association with the necessary physiologically acceptable diluent, i.e., carrier or vehicle.

[0272] In some embodiments, anti-GPC3 antibody or its antigen-binding portion or conjugate, or any of these pharmaceutical compositions, is administered together with immunotherapy.As used herein, "immunotherapy" refers to a therapeutic strategy designed to induce or enhance the subject's own immune system to fight cancer or malignant tumors.Examples of immunotherapy include, but are not limited to, antibodies such as checkpoint inhibitors.

[0273] In some embodiments, the immunotherapy involves administration of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PL-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2 and A2aR. In some embodiments, the immune checkpoint inhibitor includes agents that inhibit CTLA-4, PD-1, PD-L1, etc. Suitable anti-CTLA-4 therapeutic agents include, for example, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, murine anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, ipilimumab, tremelimumab, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 mAbs, heavy chain anti-CTLA-4 mAbs, light chain anti-CTLA-4 mAbs, inhibitors of CTLA-4 that stimulate the costimulatory pathway, include the antibodies disclosed in PCT Publication No. WO2001 / 014424, the antibodies disclosed in PCT Publication No. WO2004 / 035607, the antibodies disclosed in US Patent Application Publication No. 2005 / 0201994, and the antibodies disclosed in Granted European Patent No. 1212422B1. Additional anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; WO01 / 14424 and WO00 / 37504; US Patent Application Publication Nos. 2002 / 0039581 and 2002 / 086014.Other anti-CTLA-4 antibodies that can be used in the methods of the disclosure include those disclosed in, for example, PCT Publication No. WO 98 / 42752; U.S. Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res, 58:5301-5304 (1998); U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

[0274] Suitable anti-PD-1 and anti-PD-L1 therapeutics include, for example, anti-PD-1 and anti-PD-L1 antibodies, human anti-PD-1 and anti-PD-L1 antibodies, murine anti-PD-1 and anti-PD-L1 antibodies, mammalian anti-PD-1 and anti-PD-L1 antibodies, humanized anti-PD-1 and anti-PD-L1 antibodies, monoclonal anti-PD-1 and anti-PD-L1 antibodies, polyclonal anti-PD-1 and anti-PD-L1 antibodies, chimeric anti-PD-1 and anti-PD-L1 antibodies, anti-PD-1 Adnectins and anti-PD-L1 Adnectins, anti-PD-1 domain antibodies and anti-PD-L1 domain antibodies, single chain anti-PD-1 mAbs and single chain anti-PD-L1 mAbs, heavy chain anti-PD-1 mAbs and heavy chain anti-PD-L1 mAbs, and light chain anti-PD-1 mAbs and light chain anti-PD-L1 mAbs. In specific embodiments, anti-PD-1 therapeutics include nivolumab, pembrolizumab, pidilizumab, MEDI0680, and combinations thereof. In other specific embodiments, anti-PD-L1 therapeutics include atezolizumab, avelumab, BMS-936559, durvalumab (MEDI4736), MSB0010718C, and combinations thereof.

[0275] Suitable anti-PD-1 and anti-PD-L1 antibodies are also described in Topalian et al., Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy, Cancer Cell 27:450-61 (April 13, 2015), which is incorporated by reference in its entirety.

[0276] In some embodiments, the immune checkpoint inhibitor is ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi).

[0277] In some embodiments, a method is provided for improving the treatment outcome in a subject receiving immunotherapy.The method generally comprises: administering an effective amount of immunotherapy to a subject with cancer; administering a therapeutically effective amount of GPC3 binding agent or conjugate or its pharmaceutical composition to the subject, wherein the binding agent or conjugate specifically binds to GPC3+ cancer cells;Compared to administering immunotherapy alone, the treatment outcome of the subject is improved. In some embodiments, the binding agent or conjugate thereof comprises: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 100; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52, wherein the heavy and light chain framework regions are optionally modified with 1 to 8 amino acid substitutions, deletions or insertions in the framework regions.In some embodiments, the binding agent or conjugate thereof comprises: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having an amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable (VL) region having an amino acid sequence set forth in SEQ ID NO: 34; (vi) a heavy chain variable (VH) region having an amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having an amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having an amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having an amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having an amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having an amino acid sequence set forth in SEQ ID NO: 52, wherein the binding agent specifically binds to GPC3+ cancer cells. In some embodiments, the binding agent is an antibody or an antigen-binding portion thereof. In some embodiments, the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody. In some embodiments, the binding agent is an anti-GPC3 monoclonal antibody, a Fab, a Fab', a F(ab'), an Fv, a disulfide-linked Fc, an scFv, a single domain antibody, a diabody, a bispecific antibody, or a multispecific antibody conjugate.

[0278] In some embodiments, the improved treatment outcome is an objective response selected from stable disease, partial response, or complete response as determined by standard medical criteria for the cancer being treated. In some embodiments, the improved treatment outcome is a reduction in tumor burden. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0279] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. Various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to use the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0280] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure.

[0281] All patents and other publications identified are expressly incorporated herein by reference for the purpose of, for example, describing and disclosing the methodologies described in such publications that may be used in connection with the present disclosure. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0282] The present disclosure is further illustrated by the following embodiments, which should not be construed as limiting.

[0283] 1. A conjugate comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 A binder comprising: the heavy and light chain framework regions are optionally modified with 1 to 8 amino acid substitutions, deletions or insertions in said framework regions; The binding agent comprises a binding agent that specifically binds to human GPC3; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; A conjugate comprising:

[0284] 2. The binder is (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:12; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:34; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; or (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52 2. The conjugate of embodiment 1, comprising:

[0285] 3. A conjugate comprising: A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:17, each disposed within a light chain framework region; (ii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:23, each disposed within a light chain framework region; (iii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:27, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:28, each disposed within a light chain framework region; (iv) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:104, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:32, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:33, each disposed within a light chain framework region; (v) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:4, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:36, each disposed within a light chain framework region; (vi) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:41, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:42, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:43, each disposed within a light chain framework region; (vii) the VH region comprises a complementarity determining region HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:3, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:48, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises a LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:49, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:50, each disposed within a light chain framework region; or (viii) the VH region comprises complementarity determining regions HCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 55, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 6, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56, each disposed within a light chain framework region. with a binder; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; A conjugate comprising:

[0286] 4. The conjugate according to embodiment 3, wherein said framework regions are murine framework regions.

[0287] 5. The conjugate according to embodiment 3, wherein said framework regions are human framework regions.

[0288] 6. The conjugate of any one of embodiments 1 to 5, wherein the binding agent is an antibody or an antigen-binding portion thereof.

[0289] 7. The conjugate of embodiment 6, wherein said binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0290] 8. The conjugate of any of the previous embodiments, wherein said heavy chain variable region further comprises a heavy chain constant region.

[0291] 9. The conjugate of embodiment 8, wherein the heavy chain constant region is of the IgG isotype.

[0292] 10. The conjugate of embodiment 9, wherein said heavy chain constant region is an IgG1 constant region.

[0293] 11. The conjugate according to embodiment 10, wherein said IgG1 heavy chain constant region has the amino acid sequence as set forth in SEQ ID NO: 57 or 59.

[0294] 12. The conjugate of embodiment 9, wherein said heavy chain constant region is an IgG4 constant region.

[0295] 13. The conjugate according to embodiment 10 or 11, wherein said heavy chain variable and constant regions have an amino acid sequence as set forth in any one of SEQ ID NOs: 65, 66, 68, 69, 72, 73, 76, 77, 79, 80, 82, 83, 130 and 131.

[0296] 14. The conjugate of any of the previous embodiments, wherein said light chain variable region further comprises a light chain constant region.

[0297] 15. The conjugate of embodiment 14, wherein said light chain constant region is of the kappa isotype.

[0298] 16. The conjugate according to embodiment 15, wherein said kappa light chain constant region has the amino acid sequence set forth in SEQ ID NO: 61.

[0299] 17a. The conjugate of embodiment 15 or 16, wherein the light chain variable and constant regions have an amino acid sequence as set forth in any one of SEQ ID NOs: 67, 70, 71, 74, 75, 78, 81 and 84.

[0300] 17b.(i) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO:65 or 66, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO:67; (ii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 70; (iii) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 71; (iv) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 130 or 131, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 74; (v) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 72 or 73, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 75; (vi) the heavy chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 76 or 77, and the light chain variable and constant regions have the amino acid sequence set forth in SEQ ID NO: 78; (vii) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 79 or 80, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 81; or (viii) The conjugate according to any of the preceding embodiments, wherein said heavy chain variable and constant regions have the amino acid sequence as set forth in SEQ ID NO: 82 or 83, and said light chain variable and constant regions have the amino acid sequence as set forth in SEQ ID NO: 84.

[0301] 18. The conjugate of any of embodiments 1 to 17b, wherein the linker is attached to the binding agent via an interchain disulfide residue, an engineered cysteine, a glycan or modified glycan, an N-terminal residue of the binding agent, or a polyhistidine residue bound to the binding agent.

[0302] 19. The conjugate of any of the preceding embodiments, wherein the average drug loading of the conjugate is from about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

[0303] 20. The conjugate of any of the previous embodiments, wherein said binding agent is monospecific.

[0304] 21. The conjugate of any of the preceding embodiments, wherein the binding agent is bivalent.

[0305] 22. The conjugate according to any of the preceding embodiments, wherein the binding agent comprises a second binding domain and wherein the binding agent is bispecific.

[0306] 23. The conjugate of any of the previous embodiments, wherein said cytotoxic agent is selected from the group consisting of an auristatin, a camptothecin, and a calicheamicin.

[0307] 24. The conjugate of embodiment 23, wherein the cytotoxic agent is an auristatin.

[0308] 25. The conjugate of embodiment 24, wherein the cytotoxic agent is MMAE.

[0309] 26. The conjugate according to embodiment 23, wherein the cytotoxic agent is camptothecin.

[0310] 27. The conjugate according to embodiment 26, wherein said cytotoxic agent is exatecan.

[0311] 28. The conjugate of embodiment 23, wherein said cytotoxic agent is calicheamicin.

[0312] 29. The conjugate according to embodiment 28, wherein the cytotoxic agent is SN-38.

[0313] 30. The linker is selected from the group consisting of mc-VC-PAB, CL2, CL2A and (succinimid-3-yl-N)-(CH2). n 2 -C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (SEQ ID NO: 96), 2 represents an integer from 2 to 8).

[0314] 31. The conjugate according to embodiment 30, wherein the linker is mc-VC-PAB.

[0315] 32. The conjugate of embodiment 31, wherein the linker is attached to at least one molecule of MMAE.

[0316] 33. The conjugate according to embodiment 30, wherein the linker is CL2A.

[0317] 34. The conjugate according to embodiment 33, bound to at least one molecule of SN-38.

[0318] 35. The conjugate according to embodiment 30, wherein said linker is CL2.

[0319] 36. The conjugate according to embodiment 35, wherein the linker is attached to at least one molecule of SN-38.

[0320] 37. The linker is (succinimid-3-yl-N)-(CH2) n 2 -C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (SEQ ID NO: 96), 2 represents an integer of 2 to 8.

[0321] 38. The conjugate according to embodiment 37, wherein said linker is attached to at least one molecule of exatecan.

[0322] 39. A pharmaceutical composition comprising the conjugate of any of the preceding embodiments and a pharma- ceutically acceptable carrier.

[0323] 40. A nucleic acid encoding a binding agent according to any of embodiments 1 to 22.

[0324] 41. A vector comprising the nucleic acid according to embodiment 40.

[0325] 42. A cell line comprising a nucleic acid according to embodiment 41.

[0326] 43. A method for treating GPC3+ cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate according to any of embodiments 1 to 38 or a pharmaceutical composition according to embodiment 39.

[0327] 44. The method of embodiment 43, wherein the GPC3+ cancer is a cancer or malignant tumor.

[0328] 45. The method of embodiment 44, wherein the GPC3+ cancer is selected from hepatocellular carcinoma, lung cancer, such as small cell lung cancer and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, renal cell carcinoma, breast cancer (e.g., triple-negative breast cancer), melanoma, germ cell cancer (e.g., testicular), gastric cancer, sarcoma and bladder cancer.

[0329] 46. ​​The method of any of embodiments 43 to 45, further comprising administering an immunotherapy to the subject.

[0330] 47. The method of embodiment 46, wherein the immunotherapy comprises an immune checkpoint inhibitor.

[0331] 48. The method of embodiment 47, wherein the immune checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

[0332] 49. The method of embodiment 48, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab or ipilimumab.

[0333] 50. The method of any of embodiments 43 to 49, further comprising administering chemotherapy to the subject.

[0334] 51. The method of any of embodiments 43 to 50, wherein the conjugate is administered intravenously.

[0335] 52. The method of any of embodiments 43 to 51, wherein the conjugate is administered at a dose of about 0.1 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 12 mg / kg.

[0336] 53. A method for improving the treatment outcome of a subject undergoing immunotherapy and / or chemotherapy for GPC3+ cancer, comprising: administering an effective amount of immunotherapy or chemotherapy to a subject having cancer; administering to the subject a therapeutically effective amount of a conjugate according to any of embodiments 1 to 36 or a pharmaceutical composition according to embodiment 37, wherein said treatment outcome in said subject is improved as compared to administration of said immunotherapy or chemotherapy alone.

[0337] 54. The method of embodiment 53, wherein said improved treatment outcome is an objective response selected from stable disease, partial response, or complete response.

[0338] 55. The method of embodiment 53, wherein the improved treatment outcome is a reduction in tumor burden.

[0339] 56. The method of embodiment 53, wherein the improved treatment outcome is progression-free survival or disease-free survival.

[0340] 57. The method of any one of embodiments 53 to 56, wherein the immunotherapy is an immune checkpoint inhibitor.

[0341] 58. The method of embodiment 57, wherein the immune checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4.

[0342] 59. The method of embodiment 58, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab or ipilimumab.

[0343] 60. The method of any of embodiments 53 to 59, wherein the conjugate is administered intravenously.

[0344] 61. The method of any of embodiments 53 to 60, wherein the conjugate is administered at a dose of about 0.1 mg / kg to about 10 mg / kg.

[0345] 62. Use of a conjugate according to any of embodiments 1 to 38 or a pharmaceutical composition according to embodiment 37 for treating GPC3+ cancer in a subject.

[0346] 63. Use of a conjugate according to any of embodiments 1 to 38 or a pharmaceutical composition according to embodiment 37 for treating GPC3+ cancer in a subject undergoing immunotherapy or chemotherapy. EXAMPLES

[0347] Methods and Materials The following methods and materials were used in the examples below.

[0348] Antibody engineering by soft mutagenesis - A phage library was generated from soft randomization of regions on HCDR3 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO: 8) and introduction of site saturation mutations on LCDR1 (SEQ ID NO: 6) and HCDR2 (SEQ ID NO: 4) of ARD-103 antibody. CDR positions were identified by Kabat numbering. Competent bacterial TG1 cells were transformed with phagemid vectors containing scFv products. The size of the library ranged from 4E+07 to 3.2E+09. Stable variants were selected after a series of affinity driven panning and heat treatment screening. Affinity panning was performed by panning against high and low antigen concentrations to select high affinity binders. Recovered phages were packaged into polyvalent phages and subjected to heat treatment. After each panning round, the titers of recovered phages were determined and sequenced. Lead scFv variants were selected and screened by binding, aggregation and off-rate ranking by Octet. After IgG conversion, characterization of successful clones included binding ELISA, Biacore for affinity measurements and thermostability.

[0349] Binding ELISA-Nunc Immuno Maxisorp 96-well plates (ThermoFisher) were coated overnight with recombinant human GPC3 protein (R&D Systems). Plates were then washed, blocked, and incubated with test mAbs for 2 hours. Detection of bound test agents was determined upon subsequent incubation with goat anti-human Fc specific secondary antibody (Sigma) and TMB substrate.

[0350] Cell Culture - GPC3+HepG2 and HepG2-C3A hepatoma cells were obtained from ATCC (Manassas, VA) and maintained in culture according to the supplier's instructions.

[0351] Preparation of drug conjugates - ARD103-vcMMAE (ARD103-mc-vc-PAB-MMAE) and variant ARD103-vcMMAE conjugates were prepared by stochastic conjugation in sodium borate buffer (pH 7.4) at room temperature. Briefly, ARD103 (heavy chain = SEQ ID NO: 9; light chain = SEQ ID NO: 10) or variants (VH and VL tethered to IgG1 and Ig kappa constant regions, respectively, sequences listed in Table A) were reduced with TCEP (Tris, 2-carboxyethylphosphine) and then incubated with the drug linker, mc-vc-PAB-MMAE, at a payload:antibody ratio of 10:1. Excess drug linker was removed by dialysis. Conjugate purity was confirmed by size exclusion HPLC (99% monomer, <1% aggregates). Drug loading was an average of 4 as assessed by LC-MS. A representative structure of ARD103-vcMMAE is shown below (for vcMMAE conjugates of variant ARD103, use variant ARD103 instead of ARD103). [ka]

[0352] ARD103-CL2A-SN38 and variant (P1-B9, P1-D6 and P6-D11) CL2A-SN38 conjugates were prepared in PBS buffer, pH 7.4, at room temperature. Briefly, ARD103 or variant antibodies were reduced with TCEP and then incubated with the drug linker, CL2A-SN38, in a 10:1 ratio. The reaction was quenched with N-ethylmaleimide. Excess drug linker was removed by dialysis. Size-exclusion HPLC confirmed the conjugate purity (98.5% monomer, 1.5% aggregates). Drug loading, assessed by LC-MS, was an average of 8. A representative structure of ARD103-CL2A-SN38 is shown below (for CL2A-SN38 conjugates to variant ARD103 antibodies, use variant ARD103 instead of ARD103). [ka]

[0353] In vitro cytotoxicity assay - HepG2 cells were harvested with trypsin and plated in tissue culture medium at 1500 cells / well in 96-well clear flat-bottom, black-walled tissue culture plates. The next day, test compounds (ADCs prepared by serial dilution to generate 10-point dose curves) or vehicle were added. Cells were incubated for 96 hours. Cell viability was determined using CelltiterGlo (Promega, Madison, WI) according to the manufacturer's instructions. Data were graphed using Prism (GraphPad, La Jolla, CA).

[0354] Mouse xenograft study - All animal experiments were performed according to IACUC (Institutional Animal Care and Use) approved protocols in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines. For the liver cancer model, 5 million HepG2-C3A cells were implanted into the right flank of NOD / SCID mice. Tumor growth was monitored twice weekly using caliper measurements and calculated using the formula (V = 0.5a × b 2 Tumor volume was calculated using the formula: a = longest diameter and b = shortest diameter. 3 When the tumor growth rate reached 100%, the mice were treated intravenously with the test compound. The mice were monitored for tumor growth, body weight, and general health for 3 weeks after the last dose of test agent. Data were graphed using Prism (GraphPad, La Jolla, CA).

[0355] Example 1: Anti-GPC3 antibody engineering ARD103 antibody variants were generated by soft randomization of the regions on HCDR3 and LCDR3, and by introducing site saturation mutations on LCDR1 and HCDR2 of ARD103 antibody. After a series of affinity-driven panning and heat treatment screening, stable variants were selected. Figure 1 shows the effect of heat treatment on the ability of scFv to bind to recombinant hGPC3. Multivalent phages generated after successive rounds of affinity-driven panning were heated at various temperatures (25°C, 60°C, 70°C) for 5 minutes before testing their binding to hGPC3 by ELISA. The best clones were selected for off-rate ranking by Octet. In this example, phages P1-B9, P1-E6, P3-A1 and P1-C11 showed better binding ability after heat treatment compared to parent ARD-103.

[0356] Table 2 shows the amino acid changes in the CDRs of 15 lead scFvs (sequenced from constructs from phage display screening). Variants were selected after multiple rounds of panning, heat treatment testing, and ranked according to off-rates measured by Octet. Amino acid changes compared to reference ARD-103 are shown in bold. [Table 2-1] [Table 2-2]

[0357] The lead scFv was converted to IgG format by tethering the VH to an IgG1nG1m1 allotype variant constant region and the VL to an Ig kappa constant region. Not all of the clones expressed well in full-length IgG format. Table 3 shows the binding kinetics of the eight lead IgG variants of ARD-103 as determined by Biacore. The kinetic model was 1:1 binding. The analyte was recombinant hGPC3-His. The capture solution was 2-5 μg / ml antibody. The association constant (k a ) is approximately two-fold higher than the association constant of ARD-103. [Table 3-1] [Table 3-2]

[0358] Figure 2 shows the binding of the five lead IgG variants of ARD-103 (P1-B9, P1-D6, P1-G5, P3-F7 and P6-D11) to recombinant hGPC3 as determined by ELISA. As can be seen in the figure and table, the binding of the five lead antibodies to hGPC3 was similar to that shown by ARD-103. The EC50 was approximately 0.1 nM under these conditions.

[0359] The HCDR1-3, LCDR1-3, VH, VL and constant region sequences of the lead ARD103 variant in IgG format expressed in HEK293 cells are provided in Table 1.

[0360] Example 2: Activity of ARD103 and variant MAB-VCMMAE in in vitro cytotoxicity assays. The activity of the conjugates of ARD-103 antibody or lead antibody variants conjugated to vcMMAE was evaluated in an in vitro cytotoxicity assay against HepG2 cells. Cells were incubated with the ADC for 96 hours. As can be seen in Figure 3, IC50 values ​​for the variants ranged from 0.3 to 0.43 nM, and the activity of P6-D11, P1-B9 and P1-D6 was comparable to that of ARD-103. Under these conditions, the maximum cell killing of the P6-D11 and P1-B9 conjugates was similar to that of the parent ARD103-vcMMAE (30-31%) (Figure 3).

[0361] Example 3: In vivo activity of variant mAbs conjugated to ARD103 and CL2A-SN38 The antitumor efficacy of ARD103-CL2A-SN38 and CL2A-SN38 conjugates of three mAb variants (P1-B9, P1-D6 and P6-D11) and control hIgG was tested in a HepG2-C3A liver cancer xenograft model. Tumor-bearing mice (8 mice / group) were given 4 doses of 12 mg / kg ADC intravenously once every 4 days (arrows shown in Figure 4). In this model, ARD103-CL2A-SN38 and CL2A-SN38 conjugates of three mAb variants (P1-B9, P1-D6 and P6-D11) were effective in reducing tumor growth compared to vehicle or control ADC groups (p<0.001). Mice treated with the control hIgG-CL2A-SN38 ADC showed a 37% tumor growth delay by day 36, but tumors continued to grow steadily thereafter, with tumor size comparable to the vehicle group at the end of the experiment. There was durable complete regression in 8 of 8 mice treated with ARD103-CL2A-SN38 or P6-D11-CL2A-SN38. In comparison, 7 of 8 mice in the P1-D6-CL2A-SN38 group were tumor-free at the end of the experiment. Tumor growth was significantly reduced in mice treated with P1-B9-CL2A-SN38, but this effect was not sustained and tumors grew steadily again in all 8 mice. The three mAb variants (P1-B9, P1-D6 and P6-D11) had comparable K D As shown in Fig. 1, the antitumor effect of P6-D11 was superior to that of P1-D6 in this experiment as a CL2A-SN38 conjugate, and both of these variants were superior to P1-B9.

[0362] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0363] Various publications, including patents, patent application publications, and scientific literature, are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0364] [ka] [ka] [ka] SEQ ID NO:89 - Gly4Ser synthetic linker GGGGS SEQ ID NO:90 - (Gly4Ser)2 synthetic linker GGGGSGGGGS SEQ ID NO:91 - (Gly4Ser)3 synthetic linker GGGGSGGGGSGGGGS SEQ ID NO:92 - (Gly4Ser)4 synthetic linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:93 - (Gly4Ser)5 synthetic linker GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:94 - hexahistidinyl tag His His His His His SEQ ID NO:95 - cleavable peptide linker Gly-Phe-Leu-Gly SEQ ID NO:96 - cleavable peptide linker Gly-Gly-Phe-Gly SEQ ID NO:97 - cleavable peptide linker Ala-Leu-Ala-Leu

[0365] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheets, including but not limited to U.S. Patent Application No. 63 / 281,454, filed November 19, 2021, and U.S. Patent Application No. 63 / 326,061, filed March 31, 2022, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to use concepts from various patents, applications, and publications to provide further embodiments.

[0366] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure.

Claims

1. A conjugate comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 45; (ii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 52; (iii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 12; (iv) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 19; (v) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 24; (vi) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 128 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 29; (vii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 34; (viii) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 38; or (ix) A heavy chain variable (VH) region and a light chain variable (VL) region of any one of (i) to (viii). A binder comprising: the heavy and light chain framework regions are modified by 1 to 8 amino acid substitutions, deletions or insertions in said framework regions; the binding agent specifically binds to human GPC3; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; , a conjugate comprising:

2. A conjugate comprising:

1. A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:48, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:49, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:50, each disposed within a light chain framework region; (ii) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:55, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:6, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:56, each disposed within a light chain framework region; (iii) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 17, each disposed within a light chain framework region; (iv) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 23, each disposed within a light chain framework region; (v) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:27, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:28, each disposed within a light chain framework region; (vi) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 104, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 32, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 33, each disposed within a light chain framework region; (vii) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO:3, HCDR2 having the amino acid sequence set forth in SEQ ID NO:4, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO:16, LCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:36, each disposed within a light chain framework region; or (viii) the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 41, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 5, each disposed within a heavy chain framework region; and the VL region comprises LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 42, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43, each disposed within a light chain framework region. a binder; at least one linker attached to the binding agent; at least one cytotoxic agent attached to each linker; , a conjugate comprising:

3. The conjugate of claim 2 , wherein the framework regions are murine framework regions.

4. The conjugate of claim 2 , wherein the framework regions are human framework regions.

5. The conjugate of claim 1 , wherein the binding agent is an antibody or an antigen-binding portion thereof.

6. 6. The conjugate of claim 5, wherein the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

7. 5. The conjugate of claim 1, wherein the heavy chain variable region further comprises: (i) a heavy chain constant region; (ii) a heavy chain constant region of an IgG isotype; (iii) a heavy chain constant region that is an IgG1 constant region; (iv) a heavy chain constant region that is an IgG1 heavy chain constant region and has the amino acid sequence set forth in SEQ ID NO: 57 or 59; or (v) a heavy chain constant region that is an IgG4 constant region.

8. 8. The conjugate of claim 7, wherein the heavy chain variable and constant regions have the amino acid sequence set forth in any one of SEQ ID NOs: 65, 66, 68, 69, 72, 73, 76, 77, 79, 80, 82, 83, 130 and 131.

9. 5. The conjugate of claim 1, wherein the light chain variable region further comprises: (i) a light chain constant region; (ii) a light chain constant region of a kappa isotype; (iii) a light chain constant region of a kappa isotype, wherein the kappa light chain constant region has the amino acid sequence set forth in SEQ ID NO: 61; or (iv) a light chain constant region, wherein the light chain variable and constant regions have the amino acid sequence set forth in any one of SEQ ID NOs: 67, 70, 71, 74, 75, 78, 81 and 84.

10. (i) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 65 or 66, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 67; (ii) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 70; (iii) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 68 or 69, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 71; (iv) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 130 or 131, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 74; (v) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 72 or 73, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 75; (vi) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 76 or 77, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 78; (vii) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 79 or 80, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 81; or (viii) the heavy chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO: 82 or 83, and the light chain variable and constant regions have the amino acid sequences set forth in SEQ ID NO:

84. The conjugate of claim 8.

11. The conjugate of any one of claims 1 to 4, wherein the linker is attached to the binder via an interchain disulfide residue, an engineered cysteine, a glycan or modified glycan, an N-terminal residue of the binder, or a polyhistidine residue attached to the binder.

12. 5. The conjugate of any of claims 1 to 4, wherein the average drug loading of the conjugate is from about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

13. 5. The conjugate of claim 1, wherein the binding agent is (i) monospecific; (ii) bivalent; or (iii) comprises a second binding domain, such that the binding agent is bispecific.

14. 5. The conjugate of claim 1, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a camptothecin, a duocarmycin, and a calicheamicin.

15. 15. The conjugate of claim 14, wherein the cytotoxic agent is MMAE, exatecan, or SN-38.

16. The linker is selected from mc-VC-PAB, CL2, CL2A, and (succinimide-3-yl-N)-(CH ２ ) ｎ ２ -C(=O)-Gly-Gly-Phe-Gly-NH-CH ２ -O-CH ２ -(C=O)-(in the formula, n ２ The conjugate according to any one of claims 1 to 4, wherein R is selected from the group consisting of R, R, and R; (i) the linker is mc-VC-PAB; (ii) the linker is mc-VC-PAB and is attached to at least one molecule of MMAE; (iii) the linker is CL2A; (iv) the linker is CL2A and is attached to at least one molecule of SN-38; (v) the linker is CL2; (vi) the linker is CL2 and is attached to at least one molecule of SN-38; (vii) the linker is (succinimide-3-yl-N)-(CH 2 ) n 2 -C(═O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 -(C═O)- (wherein n 2 The conjugate of claim 16, wherein the linker is (succinimide-3-yl-N)-(CH 2 ) n 2 -C(═O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 -(C═O)- (wherein n 2 represents an integer from 2 to 8), or (viii) the linker is (succinimide-3-yl-N)-(CH 2 ) n 2 -C(═O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 -(C═O)- (wherein n 2 represents an integer from 2 to 8), and the linker is attached to at least one molecule of exatecan.

18. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding a binding agent according to any one of claims 1 to 4.

20. A vector comprising the nucleic acid of claim 19.

21. 20. A cell line comprising the nucleic acid of claim 19.

22. 10. A composition for use in a method of treating GPC3+ cancer, said composition comprising a conjugate according to any one of claims 1 to 4, said method comprising administering a therapeutically effective amount of said composition to a subject in need of treatment.

23. 23. The composition of claim 22, wherein the GPC3+ cancer is a cancer or malignant tumor.

24. 24. The composition of claim 23, wherein the GPC3+ cancer is selected from hepatocellular carcinoma, lung cancer such as small cell lung cancer, squamous cell lung cancer, and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, ovarian cancer, renal cell carcinoma, breast cancer, melanoma, germ cell cancer (e.g., testicular), thyroid cancer, vulvar cancer, gastric cancer, sarcoma, and bladder cancer.

25. The composition described in claim 22, characterized in that the composition is administered to the subject in combination with immunotherapy.

26. 26. The composition of claim 25, wherein the immunotherapy comprises an immune checkpoint inhibitor.

27. 27. The composition of claim 26, wherein the immune checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

28. 28. The composition of claim 27, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

29. The composition of claim 22, wherein the composition is administered to the subject in combination with chemotherapy.

30. 23. The composition of claim 22, wherein the conjugate is administered intravenously, administered at a dose of about 0.1 mg / kg to about 12 mg / kg, or any combination thereof.

31. 10. A composition for use in a method for improving the treatment outcome of a subject undergoing immunotherapy and / or chemotherapy of a GPC3+ cancer, said composition comprising a conjugate according to any one of claims 1 to 4, said method comprising: administering an effective amount of immunotherapy or chemotherapy to said subject with cancer; administering a therapeutically effective amount of the composition to the subject; The composition improves the treatment outcome in the subject compared to administration of the immunotherapy or chemotherapy alone.

32. 32. The composition of claim 31, wherein the improved treatment outcome is (i) an objective response selected from stable disease, partial response, or complete response; (ii) a reduction in tumor burden; or (iii) progression-free survival or disease-free survival.

33. 32. The composition of claim 31, wherein the immunotherapy is an immune checkpoint inhibitor.

34. 34. The composition of claim 33, wherein the immune checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4.

35. 35. The composition of claim 34, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

36. 32. The composition of claim 31, wherein the conjugate is administered intravenously, administered at a dose of about 0.1 mg / kg to about 10 mg / kg, or any combination thereof.

37. 1. A composition for use in treating a GPC3+ cancer in a subject, the composition comprising a conjugate, the conjugate comprising: (1) A binder, a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:45; or A heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:

52. Including, the binding agent specifically binds to human GPC3; (2) at least one linker attached to the binding agent; (3) at least one cytotoxic agent attached to each linker; A composition comprising:

38. 1. A composition for use in treating a GPC3+ cancer in a subject receiving immunotherapy or chemotherapy, the composition comprising a conjugate, the conjugate comprising: (1) A binder, a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO:44 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:45; or A heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 51 and a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO:

52. Including, the binding agent specifically binds to human GPC3; (2) at least one linker attached to the binding agent; (3) at least one cytotoxic agent attached to each linker; A composition comprising:

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