GPC3-targeted molecules and uses thereof

Abstract

Various embodiments of the invention provide binding agents for glypican-3 (GPC3), including antibodies or antigen-binding fragments thereof that bind to GPC3 and antigen recognizing receptors (e.g., CARs) that target GPC3 including cells that express GPC3. Embodiments of the i n vention also provide cells comprising such binding agents such as CAR T-cells, therapeutic compositions comprising such cells and methods of using one or more of the binding agents, cells or therapeutic compositions for the treatment of diseases or disorders associated with GPC3 expression such as various neoplastic diseases or disorders (e.g., hepatocellular carcinoma). Various embodiments of the invention also provide methods and systems for treati ng hepatocellular carcinoma (HCC) utilizing a combi nation of radiofrequency (RF) ablation and GPC3-targeted cell therapy. In certain embodiments, immune modulators, such as checkpoint inhibitors, may be co-administered to potentiate immune responses. Additionally, compositions and kits comprising RF ablation devices and GPC3-targeted cell therapy formulations are provided.

Description

Attorney Docket No.: ABH-01225GPC3-TARGETED MOLECULES AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 738,605, filed December 24, 2024, and U.S. Provisional Application No. 63 / 752,265, filed January 31, 2025, the contents of each of which are herein incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING XML

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. Said XML file, created on December 15, 2025, is named ABH-01225_SL.xml and is 180,422 bytes in size.TECHNICAL FIELD

[0003] Embodiments of the invention relate to molecules that target glypican-3 (GPC3), cells comprising the molecules and methods for treating GPC3-related diseases or disorders using such molecules and cells. Embodiments of the invention also provide a combination of ablative therapies and immunotherapies for the treatment of GPC3-related diseases or disorders, such as liver cancer. More specifically, embodiments of the invention provide a combination of radiofrequency (RF) ablation therapies and GPC3-based cell therapies for the treatment of hepatocellular carcinoma (HCC).BACKGROUND

[0004] Cell -based immunotherapy has curative potential for the treatment of cancer. T-cells and other immune cells may be modified to target tumor antigens through the introduction of genetic material coding for artificial or synthetic receptors for antigen, termed Chimeric Antigen Receptors (CARs), specific to selected antigens. Targeted T-cell therapy using CARs has shown recent clinical success in treating hematologic malignancies but has not seen widespread application for solid tumors due to a lack of or limited number of common target tumor markers (e.g., antigens found only on solid tumors cells and not cells of healthy organ tissue).1FH13240754.1Attorney Docket No.: ABH-01225

[0005] One such potential tumor marker is GPC3. It is widely expressed in the placenta and fetal tissues, but its expression is significantly reduced in adult organs. However, high expression levels of GPC3 have been found in hepatocellular carcinoma (HCC) cells compared to normal liver and non-neoplastic liver lesions.

[0006] HCC is the most common type of primary liver cancer in adults and is currently the most common cause of death in people with cirrhosis. It is the third leading cause of cancer-related deaths worldwide. While there are a number of treatments for HCC, the usual outcome is poor because only 10-20% of hepatocellular carcinomas can be removed completely using surgery. If the cancer cannot be completely removed, the disease is usually deadly within 3 to 6 months. While there are alternative treatments to surgery such as various ablative therapies, there are limitations on their clinical efficacy. In particular, thermally ablative therapies, such as radiofrequency (RF) ablation, treat a tumor at a macroscopic level by the application of radio frequency energy to generate heat at a target tumor site and cause localized tumor destruction. These therapies cannot target the tumor at a microscopic or cellular level, often resulting in the survival of residual cancer cells and the subsequent tumor recurrence.

[0007] Owing to the poor prognosis for HCC with current therapies and since GPC3 is a promising tumor marker and a potential molecular target for therapeutic intervention in HCC, there is a need for GPC3-targeted molecules, e.g., CARs or antibodies, for the treatment of HCC or any solid tumors or other tumorous tissue with high expression levels of GPC3. More specifically, there is a need for GPC3-targeted molecules that may be used alone or in combination with existing therapies. There is also a need for improvements to ablative therapies in the form of supplemental or adjunct therapies to ensure that post-ablation surviving tumor cells are targeted and destroyed before they can cause a recurrence or relapse of the disease at the tumor site or elsewhere in the patient’s body (e.g., through the process of metastasis).SUMMARY

[0008] Various embodiments of the invention provide GPC3-targeted molecules and binding agents (e.g., antibodies, antigen-binding fragments, chimeric antigen receptor (CAR)s), cells comprising the GPC3-targeted molecules and binding agents, and compositions and uses of the cells and GPC3-targeted molecules and binding agents, e.g., for treating a GPC3-related disease or disorder. More particularly, many embodiments provide 2FH13240754.1Attorney Docket No.: ABH-01225binding agents of GPC3-targeted CARs and anti-GPC3 antibodies or antigen-binding fragments, cells comprising the GPC3-targeted CARs, and compositions and uses of the cells and binding agents, e.g., for treating a GPC3-related disease or disorder. In some embodiments, the GPC3-targeted CARs and cells comprising the GPC3-targeted CARs are configured to have improved expansion and proliferation properties as compared to those known in the art. Various embodiments of the invention are particularly useful for the treatment of various diseases or conditions where the diseased tissue has high expression levels of GPC3 including, for example, hepatocellular carcinoma or other neoplastic disease or condition (e.g., cancer).

[0009] Various embodiments of the invention also provide a combination of ablative therapies and immunotherapies for the treatment of a GPC3-related disease or disorder, such as liver cancer. More specifically, some embodiments provide a combination of a radiofrequency (RF) ablation therapy and a GPC3-based cell therapy for the treatment of Hepatocellular carcinoma (HCC). In some embodiments, the GPC3-based cell therapy comprises one or more GPC3-targeted molecules and binding agents (e.g., antibodies, antigen-binding fragments, chimeric antigen receptor (CAR)s), or cells comprising the GPC3-targeted molecules and binding agents including humanized binding agents as described herein.

[0010] In a first aspect, embodiments of the invention provide a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain that binds to GPC3, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 358, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 133, and the VL comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 136, wherein: (a) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence with at least about 95% identity to the amino 3FH13240754.1Attorney Docket No.: ABH-01225acid sequence of SEQ ID NO: 360; (b) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 361; or (c) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 362.

[0011] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360; the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361; or the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

[0012] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

[0013] In some embodiments, the extracellular domain comprises a Fv, a scFv, a Fab, or a F(ab)2. In some embodiments, the extracellular domain comprises a scFv. In some embodiments, the extracellular domain comprises a linker between the VH and the VL. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH is positioned N-terminal to the VL. In some embodiments, the VLIS positioned N-terminal to the VH. In some embodiments, the extracellular domain comprises an scFv comprising the amino acid sequence of any one of SEQ ID NOs: 363-365.

[0014] In some embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X404FH13240754.1Attorney Docket No.: ABH-01225polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide, or any combination thereof. In some embodiments, the transmembrane domain comprises a CD28 polypeptide. In some embodiments, the CD28 polypeptide comprises the amino acid sequence set forth in amino acids 154 to 179 of SEQ ID NO: 308.

[0015] In some embodiments, the intracellular domain comprises a CD3^ polypeptide. In some embodiments, the CD3^ polypeptide is a modified CD3^ polypeptide. In some embodiments, the modified CD3^ polypeptide comprises a native IT AMI, an ITAM2 variant comprising two loss-of-function mutations, and an ITAM3 comprising two loss-of-function mutations. In some embodiments, the native IT AMI comprises the amino acid sequence of SEQ ID NO: 313. In some embodiments, the ITAM2 variant comprises the amino acid sequence of SEQ ID NO: 319. In some embodiments, the ITAM3 variant comprises the amino acid sequence of SEQ ID NO: 323. In some embodiments, the modified CD3^ polypeptide comprises the amino acid sequence of SEQ ID NO: 325.

[0016] In some embodiments, the intracellular domain comprises at least one co-stimulatory signaling region which may comprise at least one intracellular signaling domain of a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD28, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD8, CD4, B2C, CD80, CD86, DAP10, DAP12, MyD88, BTNL3NKG2D or combinations thereof. In some embodiments, the co-stimulatory molecule is CD28. In some embodiments, the at least one co-stimulatory signaling region comprises amino acids 180 to 220 of SEQ ID NO: 308.

[0017] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 367. In some embodiments, the CAR comprises a spacer between the extracellular domain that binds to GPC3 and the transmembrane domain. In some embodiments, the spacer comprises the amino acid sequence RAA. In some embodiments, the CAR comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the CAR comprises a self-cleaving P2A peptide. In some embodiments, the self-cleaving P2A peptide comprises the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the self-cleaving P2A peptide is at the C-terminus of the intracellular domain. In some embodiments, the CAR comprises a TGFf) dominant negative receptor (DNR). In some embodiments, the TGF DNR comprises 5FH13240754.1Attorney Docket No.: ABH-01225the amino acid sequence of SEQ ID NO: 366. In some embodiments, the CAR comprises a self-cleaving P2A peptide. In some embodiments, the self-cleaving P2A peptide comprises the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the selfcleaving P2A peptide is at the C-terminus of the intracellular domain and links the TGF DNR to the intracellular domain. In some embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 368-370.

[0018] In some embodiments, the CAR is expressed from a vector such as a viral vector which may correspond in various embodiments to a retroviral vector or a lentiviral vector.

[0019] In some embodiments, the CAR inhibits growth of GPC3-expressing cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the GPC3-expressing cells are Huh7 cells. In some embodiments, the CAR prolongs proliferation of T cells transduced with the CAR in a subject by at least 7, at least 14, at least 21, or at least 28 days. In some embodiments, the CAR induces proliferation of T cells transduced with the CAR in a subject for at least 7, at least 14, at least 21, at least 28, at least 35, at least 42, or at least 49 days. In some embodiments, the CAR induces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold higher expansion of T cells transduced with the CAR compared to T-cells not transduced with a CAR. In some embodiments, the CAR induces secretion of IFNy in a subject. In some embodiments, the CAR increases secretion of IFNy in a subject by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold.

[0020] In a second aspect, embodiments of the invention provide a nucleic acid encoding a CAR disclosed herein. In some embodiments, the nucleic acid comprises a promoter operably linked to the sequence encoding the CAR. In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is an elongation factor (EF)-l promoter, a cytomegalovirus immediate-early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter or a Ubiquitin C promoter. In some embodiments, the endogenous promoter is a TCR alpha promoter, a TCR beta promoter or a beta 2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a NF AT transcri ptional response element6FH13240754.1Attorney Docket No.: ABH-01225(TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4-1BB promoter, a PD1 promoter or a LAG3 promoter.

[0021] In another aspect, embodiments of the invention provide a vector comprising a nucleic acid disclosed herein. According to some embodiments, the vector is a viral vector which may correspond to a retroviral vector or a lentiviral vector.

[0022] In yet another aspect, embodiments of the invention provide a cell comprising a CAR disclosed herein, a nucleic acid disclosed herein, or a vector disclosed herein. In some embodiments, the cell is transduced with the CAR, the nucleic acid, or the vector. In some embodiments, the CAR is constitutively expressed on the surface of the cell. In some embodiments, the cell is an immunoresponsive cell or an immune effector cell. In some embodiments, the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. In some embodiments, the cell is a T cell, a B cell, a Natural Killer (NK) cell, a macrophage, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a lymphokine activated killer (LAK) cell, a stem cell from which a lymphoid cell may be differentiated, a stem cell from which a myeloid cell may be differentiated or a combination thereof. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell, a mucosal associated invariant T cell, a af> T cell or a y5 T cell. In some embodiments, the cell is a NK cell. In some embodiments, the NK cell is derived from a stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryoid stem cell or an induced pluripotent stem cell.

[0023] In still yet another aspect, embodiments of the invention provide a binding agent, or a fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 358, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 133, and the VL comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 136, wherein: (a) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid 7FH13240754.1Attorney Docket No.: ABH-01225sequence of SEQ ID NO: 360, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 360; (b) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 361; or (c) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 362.

[0024] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360; the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361; or the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

[0025] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361.1n some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

[0026] In some embodiments, the binding agent or fragment thereof specifically binds GPC3. In some embodiments, the binding agent or fragment thereof is or comprises an antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is monoclonal. In some embodiments, the antibody or antigen-binding fragment is a chimeric, a humanized, or a human antibody or antigen-binding fragment. In some embodiments, the binding agent or fragment thereof is an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises a Fv, a scFv, a Fab, or a F(ab)2. In 8FH13240754.1Attorney Docket No.: ABH-01225some embodiments, the antigen-binding fragment comprises a scFv. In some embodiments, the binding agent or fragment thereof comprises a linker between the VH and the VL. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH is positioned N-terminal to the VL. In some embodiments, the VL is positioned N-terminal to the VH. In some embodiments, the binding agent or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 363-365.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises an IgA, IgG, IgM, IgE, or IgD constant domain. In some embodiments, the antibody or antigenbinding fragment comprises an IgG constant domain. In some embodiments, the antibody or antigen-binding fragment comprises an IgGl, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the antibody or antigen-binding fragment comprises an IgGl constant domain. In some embodiments, the antibody or antigen-binding fragment comprises an IgGl Fc region having an amino acid sequence of SEQ ID NO: 357. In some embodiments, the binding agent or fragment thereof binds to GPC3 with a dissociation constant (KD) of less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, or less than about 6 nM.

[0028] In yet another aspect, embodiments of the invention provide a nucleic acid that encodes a binding agent or fragment thereof disclosed herein. In some embodiments, the nucleic acid comprises a first polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 373, and a second polynucleotide comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 374-376. In some embodiments, the nucleic acid comprises a nucleic acid sequence of any one of SEQ ID NOs: 377-379.

[0029] In still yet another aspect, embodiments of the invention provide a vector comprising a nucleic acid disclosed herein. In some embodiments, the vector is an expression vector.

[0030] In yet another aspect, embodiments of the invention provide a host cell comprising a vector disclosed herein.

[0031] In still yet another aspect, embodiments of the invention provide a conjugate comprising a binding agent or fragment thereof disclosed herein. In some embodiments, the binding agent or fragment thereof is linked to a therapeutic agent, a detectable agent, or a diagnostic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug. In some embodiments, the conjugate is an immunoconjugate.9FH13240754.1Attorney Docket No.: ABH-01225

[0032] In yet another aspect, embodiments of the invention provide a multispecific molecule comprising a binding agent or fragment thereof disclosed herein, linked to a second functional moiety. In some embodiments, the second functional moiety has a different binding specificity than the binding agent or fragment thereof.

[0033] In still yet another aspect, embodiments of the invention provide a method of producing a binding agent or fragment thereof, the method comprising culturing a host cell disclosed herein under conditions which induce expression of the binding agent or fragment thereof in the host cell.

[0034] In yet another aspect, embodiments of the invention provide a method for detecting GPC3 in a whole cell or tissue, the method comprising: contacting a cell or tissue with a binding agent or fragment thereof disclosed herein, wherein the binding agent or fragment thereof comprises a detectable label; and determining the amount of the labeled binding agent or fragment thereof bound to the cell or tissue by measuring the amount of detectable label associated with the cell or tissue, wherein the amount of bound binding agent or fragment thereof indicates the amount of GPC3 in the cell or tissue.

[0035] In still yet another aspect, embodiments of the invention provide a composition comprising a cell disclosed herein, a binding agent or fragment thereof disclosed herein, a conjugate disclosed herein, or a multispecific molecule disclosed herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition comprises between about 1 x 105and about 5 x 108cells.

[0036] In yet another aspect, embodiments of the invention provide a method of treating a subject having a disease or disorder associated with expression of GPC3, the method comprising administering to the subject a cell disclosed herein, a binding agent or fragment thereof disclosed herein, a conjugate disclosed herein, a multispecific molecule disclosed herein, or a composition disclosed herein. In some embodiments, the disease or disorder is a neoplastic disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, or urothelial carcinoma. In some embodiments, the disease or disorder is hepatocellular carcinoma. In some embodiments, the subject is a human subject. In some embodiments, the method comprises administering to the subject a population of cells, wherein each cell is a cell disclosed herein. In some embodiments, the population of cells comprises T cells. In some embodiments, at least a portion of the T cells expand in the subject after administration in response to a 10FH13240754.1Attorney Docket No.: ABH-01225presence of GPC-3 expressing cells. In some embodiments, at least a portion of the T cells expand in the subject after administration for a period of up to about 28 days. In some embodiments, at least a portion of the T cells expand in the subject for a period of up to about 49 days. In some embodiments, at least a portion of the T cells secrete IFNy when engaged with cells expressing GPC3. In some embodiments, an amount of IFNy secreted by the at least a portion of the T cells peaks at about 14 to about 28 days after administration. In some embodiments, the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein an amount of IFNy secreted by the at least a portion of the T cells decreases in response to an amount of tumor shrinkage. In some embodiments, the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein a peak amount of IFNy secreted by the at least a portion of the T cells correlates with a negative inflection point in tumor size. The negative inflection point is the approximate time where the size of the tumor stops growing and starts to shrink.

[0037] In still yet another aspect, embodiments of the invention provide a method of inhibiting growth of GPC3-expressing cells in a subject, the method comprising administering to the subject a cell disclosed herein, a binding agent or fragment thereof disclosed herein, a conjugate disclosed herein, a multispecific molecule disclosed herein, or a composition disclosed herein. In some embodiments, the GPC3-expressing cells are cancer cells, wherein the administered cell is a T cell and comprises a CAR disclosed herein, and wherein the progression of growth of the GPC3-expressing cells has a negative inflection point at about 14 to about 21 days after administration of the cell. In some embodiments, the administration of the cell, binding agent, conjugate, multispecific molecule or composition and or a combination of one or more of these elements is configured to cause a cancerous tumor comprising the GPC3-expressing cells (a GPC3-positive tumor) to shrink in size in response to the administration of said elements. In particular embodiments, the administration of said elements is configured to cause the GPC3-positive tumor to shrink by about 90% to about 99% (as compared to its peak size, e.g. volume) within a period of about 28 to about 49 days with particular instances of about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% shrinkage, more particularly from about 90% to about 95% shrinkage occurring within a period of about 28 to about 49 days with particular instances of about 91%, about 92%, about 93%, about 94% or about 95% shrinkage. Such results are described and shown in Example 6 and FIGs.5A-5D.11FH13240754.1Attorney Docket No.: ABH-01225

[0038] In another aspect, embodiments of the invention provide a method for producing a cell comprising a CAR, the method comprising introducing into a cell a nucleic acid that encodes the CAR. In some embodiments, the method comprises a contacting the cell with a viral vector comprising the nucleic acid that encodes the CAR. Some embodiments provide a cell, a binding agent or fragment thereof, a conjugate, a multispecific molecule, or a composition for use in therapy. Some embodiments provide a cell, a binding agent or fragment thereof, a conjugate, a multi specific molecule, or a composition for use in a method for treating a subject for a disease or disorder associated with expression of GPC3. In some embodiments, the disease or disorder is a neoplastic disease or condition such as cancer. In some embodiments, the disease or disorder is hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma or urothelial carcinoma. In some embodiments, the disease or disorder is hepatocellular carcinoma. In some embodiments, the subject is a human subject.

[0039] In still yet another aspect, embodiments of the invention provide a method of treating a tumor, e.g., a hepatocellular carcinoma in a human or other subject comprising performing radiofrequency ablation at a target tissue site within or proximate the tumor (e.g., a hepatocellular carcinoma tumor) and administering a GPC3-targeted cell therapy in a subject. Performing radiofrequency ablation comprises delivering radiofrequency energy (or other ablative energy, e.g., microwave) to heat tissue at the target tissue site e.g., to a temperature between about 60 and about 100° C) to ablate tissue at or surrounding the target site. In some embodiments, the GPC3-targeted cell therapy is administered after performing radiofrequency ablation (e.g., the delivery of radiofrequency energy). In some embodiments, the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation, e.g., the delivery of radiofrequency energy (or other ablative therapies). In some embodiments, the GPC3-targeted cell therapy is administered intratumorally. In some embodiments, the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3. In some embodiments, the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg. In some embodiments, the radiofrequency ablation (e.g., the delivery of radio frequency energy) releases tumor-associated antigens to prime immune response. In some embodiments, the radiofrequency ablation is performed at a temperature between about 60°C and about 100°C or the delivery of radiofrequency energy heats tissue at the target tumor site to a temperature between about 60°C and about 100°C. In some embodiments, the radiofrequency ablation (e.g., the delivery 12FH13240754.1Attorney Docket No.: ABH-01225of radiofrequency energy) is performed percutaneously under ultrasound guidance. In some embodiments, the method comprises administering one or more immune checkpoint inhibitors. In some embodiments, the method comprises administering one or more cytokines to enhance immune activation. In some embodiments, the method comprises administering one or more adjuvants to enhance immune response.

[0040] In yet another aspect, embodiments of the invention provide a method of reducing tumor recurrence in a liver cancer in a subject by sequentially administering radiofrequency ablation therapy and GPC3-targeted cell therapy. In some embodiments, the GPC3-targeted cell therapy is administered after the radiofrequency ablation therapy. In some embodiments, the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation therapy. In some embodiments, the GPC3-targeted cell therapy is administered intratumorally. In some embodiments, the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3. In some embodiments, the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg. In some embodiments, the radiofrequency ablation therapy releases tumor-associated antigens to prime immune response. In some embodiments, the radiofrequency ablation therapy is performed at a temperature between about 60°C and about 100°C or the radiofrequency ablation therapy heats tissue at a target tissue to a temperature between about 60°C and about 100°C. In some embodiments, the radiofrequency ablation is performed percutaneously under ultrasound guidance. In some embodiments, the method comprises administering one or more immune checkpoint inhibitors. In some embodiments, the method comprises administering one or more cytokines to enhance immune activation. In some embodiments, the method comprises administering one or more adjuvants to enhance immune response.

[0041] In still yet another aspect, embodiments of the invention provide a method for increasing survival rate in a hepatocellular carcinoma padent, comprising administering combined radiofrequency ablation therapy and GPC3-targeted cell therapy. In some embodiments, the GPC3-targeted cell therapy is administered after the radiofrequency ablation. In some embodiments, the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation. In some embodiments, the GPC3-targeted cell therapy is administered intratumorally. In some embodiments, the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3. In some embodiments, the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg. In some embodiments, the radiofrequency ablation releases tumor-associated 13FH13240754.1Attorney Docket No.: ABH-01225antigens to prime an immune response in the subject. In some embodiments, the radiofrequency ablation is performed by the delivery of radiofrequency energy to a target tissue site (e.g., within or proximate a selected tumor) to heat tissue at the site to a temperature between about 60°C and about 100°C. In some embodiments, the radiofrequency ablation is performed percutaneously under ultrasound guidance. In some embodiments, the method comprises administering one or more immune checkpoint inhibitors. In some embodiments, the method comprises administering one or more cytokines to enhance immune activation in the subject. In some embodiments, the method comprises administering one or more adjuvants to enhance immune response in the subject.

[0042] In yet another aspect, embodiments of the invention provide a method for inducing systemic anti-tumor immunity in a hepatocellular carcinoma patient, comprising administering combined radiofrequency ablation therapy and GPC3-targeted cell therapy. In some embodiments, the GPC3-targeted cell therapy is administered after the radiofrequency ablation. In some embodiments, the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation. In some embodiments, the GPC3-targeted cell therapy is administered intratumorally. In some embodiments, the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3. In some embodiments, the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg. In these and related embodiments of the combination radiofrequency ablation and GPC3-targeted cell therapy, the dose of CAR T-cells can be selected based on one or more of the following factors : i) the size of the ablated volume of tissue including its relation to the size of the tumor treated; ii) the period of time after ablation that the cells are administered (e.g., hours, days etc.); iii) whether the treatment is a first course or that for tumor recurrence or otherwise part of a cycle of treatments ; iv) the order of treatment e.g., whether the RF ablation is performed first or vice versa; and, v) whether the cells are administered systemically or intratumorally (e.g., the latter may allow for lower doses of cells). In some embodiments, the radiofrequency ablation therapy is configured or performed in a manner to release or optimize the release of tumor-associated antigens to prime immune response. In one or more implementations, this can be achieved by one or more the following: i) control of the temperature at the target ablation site (e.g., particular temperatures can be selected which necrose or otherwise destroy tumorous tissue but minimize the thermal degradation of subsequently released TAAs), ii) control of the delivery of RF energy at the target site to produce a temperature gradient within the selected tumor such that the more 14FH13240754.1Attorney Docket No.: ABH-01225central portions of the tumor are heated to a higher temperature while tissue at the tissue margins is heated to a lower temperature, while still adequate to kill necrose cancers cells and minimize the thermal degradation of TA As; and iii) control of the size of the ablated volume of tissue, e.g., such that surrounding tissue (e.g., beyond the tumor or selected healthy tissue margin to also be ablated) is not ablated or minimally impacted to minimize thermal degradation of released TAAs). In some embodiments, the radiofrequency ablation is performed by the delivery of radiofrequency energy to a target tissue site (e.g., within or proximate a selected tumor) to heat tissue at the site to a temperature between about 60°C and about 100°C. In some embodiments, the radiofrequency ablation is performed percutaneously under ultrasound guidance. In some embodiments, the method comprises administering one or more immune checkpoint inhibitors. In some embodiments, the method comprises administering one or more cytokines to enhance immune activation. In some embodiments, the method comprises administering one or more adjuvants to enhance immune response.

[0043] In these and related embodiments, the GPC3-targeted cell therapy comprises a humanized GPC3-targeted molecule or a cell having a humanized GPC3-targeted molecule. In these and related embodiments, the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).

[0044] In still yet another aspect, embodiments of the invention provide a pharmaceutical composition comprising GPC3-targeted immune cells for use in combination with radiofrequency ablation therapy. In some embodiments, the pharmaceutical composition comprises immune modulators. In some embodiments, the GPC3-targeted immune cells comprise a humanized GPC3-targeted molecule. In some embodiments, the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).

[0045] In yet another aspect, embodiments of the invention provide a kit comprising radiofrequency ablation equipment and GPC 3 -targeted cell therapy formulations.

[0046] In still yet another aspect, embodiments of the invention provide a kit comprising formulations for radiofrequency ablation and immune cell therapy targeting GPC-3.

[0047] In yet another aspect, embodiments of the invention provide a use of radiofrequency ablation and GPC3-targeted cell therapy for treating advanced-stage liver cancer.15FH13240754.1Attorney Docket No.: ABH-01225

[0048] In still yet another aspect, embodiments of the invention provide a therapeutic regimen combining radiofrequency ablation and GPC3-targeted cell therapy for liver cancer.

[0049] In these and related embodiments, the GPC3-targeted cell therapy comprises a humanized GPC3-targeted molecule or a cell having a humanized GPC3-targeted molecule. In these and related embodiments, the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).

[0050] Elements described with respect to some aspects or embodiments of the present specification are also contemplated with respect to other aspects or embodiments of the present specification. Moreover, recitation of claim elements in connection with a particular independent claim support recitation of such elements in connection with other independent claims. Also, throughout the disclosed embodiments and claims, where compositions or methods are described as having, including, or comprising specific elements, compositions and methods that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIGs. 1A-1B are graphs that depict the binding affinity of exemplified scFv-Fc fusion proteins of 3 humanized GPC3 binding agents (GPC3-8-hl -scFv-Fc, GPC3-8-h2-scFv-Fc, and GPC3-8-h3-scFv-Fc) and a murine parental GPC3 binding agent (GPC3-8-scFv-Fc) through results of a surface plasmon resonance (SPR) assay (FIG. 1A) and a flow cytometry (FACS) assay (FIG. IB).

[0052] FIG. 2 is a graph that depicts the binding affinity of exemplified CARs to recombinant human GPC3 through results of a flow cytometry (FACS) assay.

[0053] FIG. 3 is a graph that depicts the in vitro cytotoxicity of GPC3-targeted CAR T cells comprising the exemplified humanized or murine CARs against Huh-7D12 cells.

[0054] FIG. 4 is a schematic that depicts an experimental plan for the assessment of in vivo antitumor efficacy of GPC3-targeted CAR T cells comprising the exemplified humanized or murine CARs.

[0055] FIGs. 5A-5D are graphs that depict an assessment of the in vivo antitumor efficacy of GPC3-targeted CAR T cells comprising the exemplified humanized or murine CARs against subcutaneously implanted Huh-7D12 tumor cells in mice. Tumor size measurements in mice treated with parental murine GPC3-8 TGF DNR (FIG. 5A),16FH13240754.1Attorney Docket No.: ABH-01225humanized GPC3-8-hl TGF(3 DNR (FIG. 5B), humanized GPC3-8-h2 TGF(3 DNR (FIG. 5C), and humanized GPC3-8-h3 TGF DNR (FIG. 5D) are shown. DNR = dominant negative receptor.

[0056] FIG. 6 is a graph that depicts probability of survival in the in vivo antitumor efficacy assessment.

[0057] FIGs. 7A-7D are graphs that depict an assessment of the T cell expansion of GPC3-targeted CAR T cells comprising the exemplified humanized or murine CARs in treated mice. T cell number measurements in mice treated with parental murine GPC3-8 TGFf) DNR (FIG. 7A), humanized GPC3-8-hl TGFf) DNR (FIG. 7B), humanized GPC3-8-h2 TGFf) DNR (FIG. 7C), and humanized GPC3-8-h3 TGF DNR (FIG. 7D) are presented.

[0058] FIGs. 8A-8D are graphs that depict an assessment of in vivo IFNy secretion in mice treated with parental murine GPC3-8 TGF DNR (FIG. 8A), humanized GPC3-8-hl TGF[> DNR (FIG. 8B), humanized GPC3-8-h2 TGFf) DNR (FIG. 8C), and humanized GPC3-8-h3 TGFf) DNR (FIG. 8D).DETAILED DESCRIPTION

[0059] Various embodiments of the invention provide molecules that target GPC3 (referred to as “GPC3-targeted molecules”). In some embodiments, the molecule is a binding agent or a fragment thereof (e.g., an antibody or an antigen-binding fragment thereof). In some embodiments, the molecule is an antigen recognizing receptor. In some embodiments, the antigen recognizing receptor is a chimeric antigen receptor (CAR). Many embodiments of the invention also provide cells comprising such GPC3-targeted molecules. According to one or more embodiments, the cells can be immunoresponsive cells and / or immune effector cells, e.g., genetically modified immunoresponsive cells (e.g., T cells or NK cells), or pluripotent stem cells that can differentiate into immunoresponsive cells and / or immune effector cells.Incorporation by Reference

[0060] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosure of which are incorporated by reference herein in their entireties for all purposes.Definitions17FH13240754.1Attorney Docket No.: ABH-01225

[0061] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the presently disclosed subject matter belongs.

[0062] As used herein, the term “about” or “approximately” refers to within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0063] As used herein, the term “immunoresponsive cell” refers to a cell that functions in an immune response or a progenitor, or progeny thereof. In some embodiments, the immunoresponsive cell is a cell of lymphoid lineage. Non-limiting examples of cells of lymphoid lineage include T cells, Natural Killer (NK) cells, B cells, and stem cells from which lymphoid cells may be differentiated. In some embodiments, the immunoresponsive cell is a cell of myeloid lineage.

[0064] As used herein, the term “activates an immunoresponsive cell” refers to induction of signal transduction or changes in protein expression in the cell resulting in initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibition motifs (IT AMs) a signal transduction cascade is produced. In some embodiments, when a CAR binds to an antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3y / 5 / e / ^, etc.). This clustering of membrane bound signaling molecules allows for IT AM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation in turn initiates a T cell activation pathway ultimately activating transcription factors, such as NF-KB and AP-1. These transcription factors induce global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response.

[0065] As used herein, the term “stimulates an immunoresponsive cell” refers to a signal that results in a robust and sustained immune response. In some embodiments, this occurs 18FH13240754.1Attorney Docket No.: ABH-01225after immunoresponsive cell (e.g., T cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, 4-1BB, 0X40, CD40 and ICOS. Receiving multiple stimulatory signals can be important to mount a robust and long-term T-cell mediated immune response. T cells can quickly become inhibited and unresponsive to antigen. While the effects of these co- stimulatory signals may vary, they generally result in increased gene expression in order to generate long lived, proliferative, and anti-apoptotic T cells that robustly respond to antigen for complete and sustained eradication.

[0066] The term “antibody” covers, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full length heavy and / or light chains. The disclosed embodiments also include antibody fragments (and / or polypeptides that comprise antibody fragments) that retain a5 integrin binding characteristics. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv , single chain antibody molecule, dual variable region antibody, single variable region antibody, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., VHH, nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In general terms, a variable region domain may be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable regions. For example, provided herein are tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer. Thus, for example, the variable region domain may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers that bind a5 integrin. If desired, the VH and VL chains may be covalently coupled either directly or through a linker to form a single chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs can be obtained by constructing polynucleotides that encode the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using 19FH13240754.1Attorney Docket No.: ABH-01225mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). The antibodies or antigen-binding fragments thereof, in some embodiments, comprise a light chain and / or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, antibodies can include epitopebinding fragments of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule. Antibodies can be antagonistic antibodies or agonistic antibodies.

[0067] As used herein, “affinity” refers to the strength of the sum total of non-covalent interactions between a particular binding agent (e.g., a receptor), and / or a binding site thereof, with a binding target (e.g., a ligand). Unless indicated otherwise, as used herein, “binding affinity” refers to a 1 : 1 interaction between a binding agent and a binding target thereof (e.g. , a receptor and ligand pair). Those of skill in the art appreciate that a change in affinity can be described by comparison to a reference (e.g., increased or decreased relative to a reference), or can be described numerically. Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and / or equilibrium association constant (KA). The KD is the quotient of kOff / kOn, whereas KA is the quotient of kon / koff, where konrefers to the association rate constant of, e.g., antibody with antigen, and kon refers to the dissociation of, e.g., antibody from antigen. konand kOff can be determined by techniques known to those of skill in the art, such as BIACORE® or KinExA.

[0068] As used herein, the term “binding” refers to a non-covalent association between or among two or more agents. “Direct” binding involves physical contact between agents; indirect binding involves physical interaction by way of physical contact with one or more 20FH13240754.1Attorney Docket No.: ABH-01225intermediate agents. Binding between two or more agents can occur and / or be assessed in any of a variety of contexts, including where interacting agents are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier agent and / or in a biological system or cell).

[0069] As used herein, the term “monospecific antibody” refers to an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen, e.g., GPC3 e.g., human GPC3).

[0070] As used herein, the term “multispecific molecule” refers to a molecule (e.g., an antibody) that is capable of binding to at least two distinct antigenic determinants, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) binding to different antigens or to different epitopes on the same antigen. Such a multi specific molecule may have a 1+1 format. Other multispecific molecule formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a multispecific molecule comprises two antigen binding sites, each may bind to a different antigenic determinant. Such a multispecific molecule may bind to two different epitopes on the same antigen (e.g., epitopes on GPC3) or on different antigens (e.g., an epitope on GPC3 and an epitope on an antigen different from GPC3).

[0071] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0072] As used herein, the term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody’s specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced.The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by 21FH13240754.1Attorney Docket No.: ABH-01225disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0073] As used herein, the term “Fab fragment” refers to a fragment ofan antibody comprising an antigen-binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0074] As used herein, the term “F(ab')2 fragment” refers to a fragment ofan antibody containing two antigen-binding sites, generated by cleavage ofthe antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0075] As used herein, the term “Fc fragment” refers to the fragment ofan antibody comprising the constant domain of its heavy chain.

[0076] As used herein, the term “Fv fragment” refers to the fragment ofan antibody comprising the variable domains of its heavy chain and light chain.

[0077] The term “single chain variable fragment” or “scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites. The heavy chain domain and light chain domain are either joined directly or joined by a linker, which connects the N-terminus of the heavy chain domain with the C -terminus of the light chain domain, or the C-terminus of the heavy chain domain with the N-terminus of the light chain domain. As used herein, the term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. In some embodiments, the linker is a G4S linker.

[0078] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, which is provided below:GGGGSGGGGSGGGGS [SEQ ID NO : 1 ]

[0079] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, which is provided below:22FH13240754.1Attorney Docket No.: ABH-01225GGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 2 ]

[0080] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, which is provided below:GGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 3 ]

[0081] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, which is provided below:GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 4 ]

[0082] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, which are provided below.GGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCT [ SEQ ID NO : 5 ] GGCGGCGGAGGCAGCGGCGGCGGCGGCTCTGGCGGCGGCGGAAGC [ SEQ ID NO : 6 ] GGCGGAGGAGGCAGCGGCGGCGGAGGCTCTGGCGGCGGCGGAAGT [ SEQ ID NO : 7 ] GGCGGCGGAGGCAGCGGCGGCGGCGGCTCCGGCGGAGGCGGCTCT [ SEQ ID NO : 8 ] GGCGGCGGAGGCTCTGGAGGCGGAGGCAGCGGCGGCGGCGGCTCC [ SEQ ID NO : 9 ] GGCGGCGGCGGCAGCGGCGGCGGAGGTTCTGGAGGAGGCGGCAGC [ SEQ ID NO : 10 ] GGCGGCGGCGGCAGCGGCGGAGGCGGCTCTGGCGGAGGCGGCAGC [ SEQ ID NO : 11 ] GGCGGAGGCGGATCTGGCGGCGGCGGTTCAGGCGGAGGCGGCTCT [ SEQ ID NO : 12 ] GGCGGAGGCGGATCTGGCGGAGGCGGCTCTGGAGGCGGCGGCTCA [ SEQ ID NO : 13 ] GGAGGCGGCGGATCCGGCGGTGGCGGATCTGGCGGAGGCGGCAGC [ SEQ ID NO : 14 ] GGCGGAGGCGGCTCTGGAGGAGGAGGGTCCGGAGGCGGCGGCAGC [ SEQ ID NO : 15 ] GGCGGCGGCGGCAGCGGCGGAGGCGGCAGCGGCGGCGGAGGCAGC [ SEQ ID NO : 16 ] GGCGGAGGCGGCAGCGGCGGCGGCGGAAGCGGCGGCGGAGGCAGC [ SEQ ID NO : 17 ] GGCGGCGGAGGCTCTGGCGGTGGCGGAAGCGGCGGCGGCGGAAGC [ SEQ ID NO : 18 ] GGAGGCGGCGGCAGCGGCGGCGGCGGCAGTGGCGGAGGCGGCAGC [ SEQ ID NO : 19 ] GGCGGAGGCGGCAGCGGAGGAGGCGGCTCTGGCGGCGGCGGCTCA [ SEQ ID NO : 20 ] GGCGGCGGCGGCAGCGGAGGCGGCGGCAGCGGAGGAGGTGGCAGC [ SEQ ID NO : 21 ]

[0083] As used herein, “complementarity determining regions” or “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. CDRs can be identified according to a number of known numbering systems. In some embodiments, the CDRs are 23FH13240754.1Attorney Docket No.: ABH-01225identified according to the Kabat numbering system. The Kabat CDRs are based on sequence variability and are the most commonly used (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The CDRs can be identified according to the Chothia numbering system. Chothia refers to the location of the structural loops see e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The CDRs can be identified according to the AbM numbering system. The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The CDRs can be identified according to the Contact numbering system (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5: 732-745). The Contact CDRs are based on an analysis of the available complex crystal structures. The CDRs can be identified according to the ImMunoGeneTics (IMGT)® Information System. A universal numbering system has been developed and widely adopted, IMGT® Information System (Lefranc et al., Dev. Comp. Immunol. 27 (l):55-77 (2003)). IMGT® is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates.

[0084] The term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0085] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0086] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the aminoterminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, about 6, about 8 or about 10 amino acids long, at least about 1424FH13240754.1Attorney Docket No.: ABH-01225amino acids long, at least about 20, at least about 30, at least about 40 or at least about 50 amino acids long, at least about 75 amino acids long, or at least about 100, at least about 150, at least about 200, at least about 300, at least about 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and / or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.

[0087] As used herein, the term “substantially identical” or “substantially homologous” refers to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleotide sequence (for example, any of the nucleic acid sequences described herein). In some embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

[0088] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positi ve values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this specification. The polynucleotide sequences of 25FH13240754.1Attorney Docket No.: ABH-01225similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0089] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0090] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcri ption of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0091] As used herein, the term “signal sequence” or “leader sequence” refers to a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of proteins that directs their entry to the secretory pathway.

[0092] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0093] As used herein, the term “a conservati ve sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the GPC3-targeted binding comprising the amino acid sequence. Conservati ve modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include 26FH13240754.1Attorney Docket No.: ABH-01225aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function using the functional assays described herein. In some embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or a CDR region are altered.

[0094] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0095] As used herein, the term “effective amount” refers to an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In some embodiments, an effective amount can be an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount can be determined by a physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

[0096] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.27FH13240754.1Attorney Docket No.: ABH-01225

[0097] The term “treatment” refers to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0098] An “individual” or “subject” herein is a vertebrate, such as a human or a nonhuman animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of nonhuman animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys. In some embodiment, the subject is a mammal. In some embodiment, the subject is a human.

[0099] The term “GPC3 antigen” and, in particular, “GPC3” is intended to include fragments, variants (e.g., allelic variants), and derivatives of the antigen molecule, e.g., the GPC3 molecule.

[0100] The terms “comprises”, “comprising”, and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.GPC3

[0101] GPC3 is also known as glypican-3. Glypican-3 (GPC3) is a 70 kDa protein that is anchored to the cell surface via glycosylphosphatidylinositol (GPI). GPC3 is widely expressed in the placenta and fetal tissues such as liver, lung, and kidney, and it regulates morphogenesis or growth. However, its expression is significantly reduced in adult organs. On the other hand, significantly high levels of GPC3 are expressed in hepatocellular carcinoma (HCC) cells compared to normal liver and non-neoplastic liver lesions, making GPC3 a promising tumor marker and a potential molecular target for therapeutic intervention 28FH13240754.1Attorney Docket No.: ABH-01225in HCC. GPC3 is a member of the heparan sulfate (HS) proteoglycan family that consists of six members, GPC1 to GPC6, which serve as receptors or coreceptors for a number of ligand molecules, including morphogens, growth factors, adhesion, and matrix molecules, to regulate their signaling and distribution.

[0102] In some embodiments, the molecule binds to a GPC3 antigen. In some embodiments, the molecule is a binding molecule. In some embodiments, the binding molecules bind to a human GPC3 antigen. In some embodiments, the GPC3 is a human GPC3. In some embodiments, the GPC3 is a wild-type human GPC3. In some embodiments, the GPC3 antigen comprises or consists of the amino acid sequence with a Uniprot Reference No: P51654-1 (SEQ ID NO: 22), which is provided below.MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDL QVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIWRHAKN YTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPG LPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKF SKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGWEIDKYWREYILSLEELVNGM YRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKV LKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQ KAARNGMKNQFNLHELKMKGPEPWSQI IDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESG DCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNV HSPLKLLTSMAISVVCFFFLVH [SEQ ID NO : 22 ]

[0103] In some embodiments, the GPC3 antigen comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 22 or a fragment thereof.

[0104] In some embodiments, the binding molecule binds to an epitope of GPC3. In some embodiments, the epitope is within amino acids 524 to 563 of SEQ ID NO: 22. In some embodiments, the epitope is at the C-terminus of GPC3.Chimeric Antigen Receptors (CARs)

[0105] Various embodiments of the invention provide chimeric antigen receptors (CARs) that target GPC3 when it is expressed on or within the surface of a cell.29FH13240754.1Attorney Docket No.: ABH-01225

[0106] CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. CARs can be used to graft the specificity of a monoclonal antibody onto a cell, e.g., an immunoresponsive cell, e.g., a T cell or a NK cell; with transferring of their coding sequence, e.g., facilitated by retroviral or lentiviral vectors.

[0107] In some embodiments, the GPC3-targeted CAR comprises an extracellular domain that specifically binds to GPC3, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain is fused to the transmembrane domain, which is fused to the intracellular domain.

[0108] In some embodiments, the extracellular domain of the CAR comprises a GPC3-binding region, and is responsible for antigen recognition. In some embodiments, the extracellular domain further comprise a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of a cell (e.g., an immunoresponsive cell).

[0109] In some embodiments, the transmembrane domain (TD) connects the extracellular domain to the intracellular domain and resides within the cell membrane when expressed by a cell (e.g., an immunoresponsive cell).

[0110] In some embodiments, the intracellular domain transmits an activation signal to the cell (e.g., an immunoresponsive cell) after antigen recognition. In some embodiments, the intracellular domain comprises an intracellular signaling domain (ISD). In some embodiments, the intracellular domain further comprises a co-stimulatory signaling region (CSR). A “signaling domain (SD)” generally comprises immunoreceptor tyrosine-based activation motifs (IT AMs) that activate a signaling cascade when the ITAM is phosphorylated. The term “co-stimulatory signaling region (CSR)” refers to an intracellular signaling domain from a costimulatory protein receptor, such as CD28, 41BB, and ICOS, that are able to enhance the activation of an immunoresponsive cell (e.g., a T cell).

[0111] In some embodiments, the disclosed CAR is defined by the formula:SP-ED-HG-TM-CSR-SD; orSP- ED-HG-TM-SD-CSR;wherein “SP” represents an optional signal peptide,wherein “ED” represents an extracellular domain that binds to GPC3.wherein “HG” represents an optional hinge domain,wherein “TM” represents a transmembrane domain,wherein “CSR” represents one or more co-stimulatory signaling regions,wherein “SD” represents a signaling domain, and30FH13240754.1Attorney Docket No.: ABH-01225wherein represents a peptide bond or linker.

[0112] Additional CAR constructs are described, for example, in Fresnak et al., Nat. Rev. Cancer 16(9):566-81 (2016), which is incorporated by reference in its entirety for the teaching of these CAR models.

[0113] In some embodiments, the CAR can be for example (and without limitation), a TRUCK, Universal CAR, a Self-driving CAR, an Armored CAR, a Self-destruct CAR, a Conditional CAR, a Marked CAR, a TenCAR, a Dual CAR, or a sCAR.

[0114] TRUCKS (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T cell acti vation. Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.

[0115] Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0116] Self-driving CARs co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing tumor homing.

[0117] CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD-1)) using for example “Knockdown” and / or “Knockout” techniques known in the art. Exemplary “Knockdown” and “Knockout” techniques include, but are not limited to, RNA interference (RNAi) (e.g., asRNA, miRNA, shRNA, siRNA, etc.) and CRISPR interference (CRISPRi) e.g., CRISPR-Cas9). In some embodiments, CAR T cells are engineered to express a dominant-negative form of a checkpoint molecule. In some embodiments, the extracellular ligand-binding domain of the immune checkpoint molecule is fused to a transmembrane membrane in order to compete for ligand binding. For example, the extracellular ligand-binding domain of PD-1 may be fused to a CD8 transmembrane domain, thus competing for PD-1 ligand from the target cell. In some embodiments, CAR T cells are engineered to express an immune checkpoint switch receptor to exploit the inhibitory immune checkpoint ligand present on a target cell. In some embodiments, the extracellular ligand-binding domain of the immune checkpoint molecule is fused to a signaling, stimulatory, and / or co-stimulatory domain. For example, the extracellular 31FH13240754.1Attorney Docket No.: ABH-01225ligand-binding domain of PD-1 may be fused to a CD28 domain, thus providing CD28 costimulation while blocking PD-1 signaling. In some embodiments, the CAR T cells may be administered with an aptamer or a monoclonal antibody that blocks immune checkpoint signaling. In some embodiments, the CAR T cells (e.g., CAR T cell therapy) are combined with a PD-1 blockade method, such as administration with PD-1 / PD-L1 antagonistic aptamers or anti-PD-l / PD-Ll antibodies. In some embodiments, the CAR T cells and PD-1 pathway-blocking antibodies are administered conjointly. In some embodiments, the CAR T cells are engineered to express or express and secrete an immune checkpoint-blocking antibody, such as anti-PD-1 or anti-PD-Ll, or fragments thereof. In some embodiments, the CAR T cells are administered with a vector (e.g., an engineered virus) that expresses an immune checkpoint-blocking molecule described herein.

[0118] A self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of acti vation of human caspase 9 by a small-molecule dimerizer.

[0119] A conditional CAR T cell is by default unresponsive, or switched “off’, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.

[0120] Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects.

[0121] A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular costimulatory domain(s) and a CD3^ domain. TanCAR T cell activation is achieved only when target cells co-express both targets.

[0122] A dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3^ domain and the other CAR includes only the costimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.32FH13240754.1Attorney Docket No.: ABH-01225

[0123] A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0124] There are three generations of CARs. “First generation” CARs are typically composed of an extracellular antigen-binding domain (e.g., an scFv), which is fused to a transmembrane domain, which is fused to an intracellular domain. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T-cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second generation” CARs add an intracellular signaling domain from a co-stimulatory molecule (e.g., CD28, 4-1BB, ICOS, 0X40) to the intracellular domain of the CAR to provide additional signals to the cell. “Second generation” CARs comprise those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3Q. “Third generation” CARs combine multiple signaling domains to further augment potency, e.g., are made by combining multiple signaling domains, such as CD3 ^-CD28-OC40 or CD3^-CD28-4-lBB, to augment potency with stronger cytokine production and killing ability. In some embodiments, the GPC3-targeted CAR is a first-generation CAR. In some embodiments, the GPC3-targeted CAR is a second-generation CAR.

[0125] In some embodiments, the CAR comprises one signaling domain. In some embodiments, the CAR comprises one or more signaling domain (co-stimulatory signaling region). The one or more signaling domain may be a polypeptide selected from the group consisting of CD8, CD3 , CD35, CD3y, CD3a, FcyRI-y, FcyRIII-y, FcaRip, FcaRIy, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, B2C, 4-1BB (CD137), ICOS, CD27, CD288, CD80, NKp30, 0X40, and mutants thereof.Extracellular Domain of A CAR

[0126] The extracellular domain of the CARs disclosed herein generally comprise an antigen recognition domain that binds a GPC3 antigen. Such antigen-specific binding domains are typically derived from an antibody. In some embodiments, the extracellular domain of the CAR comprises or is a functional antibody fragment or derivative thereof (e.g., a single chain variable fragment (scFv) or a Fab, or any suitable antigen binding fragment of an antibody).

[0127] In some embodiments, the extracellular domain of the CAR comprises an scFv. In some embodiments, the scFv is from a monoclonal antibody (mAb). The scFv can be a human scFv, a humanized scFv, or a murine scFv. In some embodiments, the scFv is a 33FH13240754.1Attorney Docket No.: ABH-01225humanized scFv. In some embodiments, the scFv is a human scFv. The scFv can be derived from fusing the heavy chain variable region (VH) and light chain variable region (VL) of an antibody. Alternatively or additionally, the scFv may be derived from Fab’s (instead of from an antibody, e.g., obtained from Fab libraries).

[0128] In some embodiments, the extracellular domain of the CAR comprises a Fab. In some embodiments, the Fab is crosslinked. In some embodiments, the extracellular domain of the CAR comprises a F(ab)2.

[0129] Any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular domain of the CAR.

[0130] In some embodiments, the extracellular domain of the CAR binds to a GPC3 antigen (e.g., a human GPC3 antigen) with aEC50 of between about 1 ng / mL and about 2,000 ng / mL, between about 1 ng / mL and about 1,500 ng / mL, between about 1 ng / mL and about 1,000 ng / mL, between about 1 ng / mL and about 600 ng / mL, between about 1 ng / mL and about 500 ng / mL, between about 1 ng / mL and about 400 ng / mL, between about 1 ng / mL and about 300 ng / mL, between about 1 ng / mL and about 200 ng / mL, between about 1 ng / mL and about 150 ng / mL between about 1 ng / mL and about 100 ng / mL, between about 1 ng / mL and about 60 ng / mL, between about 1 ng / mL and about 50 ng / mL, between about 1 ng / mL and about 40 ng / mL, between about 4 ng / mL and about 60 ng / mL, between about 4 ng / mL and about 50 ng / mL, or between about 4 ng / mL and about 40 ng / mL. In some embodiments, the extracellular domain of the CAR binds to GPC3 antigen (e.g., a human GPC3 antigen) with a EC50 of between about 1 ng / mL and about 60 ng / mL. In some embodiments, the extracellular domain of the CAR binds to GPC3 antigen (e.g., a human GPC3 antigen) with a EC50 of between about 4 ng / mL and about 40 ng / mL.

[0131] Binding of the extracellular domain of the CAR can be confirmed by, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or a scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or 34FH13240754.1Attorney Docket No.: ABH-01225by autoradiography. In some embodiments, the GPC3-targeted extracellular domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0132] In some embodiments, the extracellular domain of the CAR comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0133] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 137; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 138. SEQ ID NO: 137 and SEQ ID NO: 138 are disclosed in Table 1.

[0134] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NOs: 132-133 and 358 are provided in Table 1.

[0135] In some embodiments, the VH comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 137. For example, the VH comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 137.

[0136] In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative35FH13240754.1Attorney Docket No.: ABH-01225modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. SEQ ID NOs: 134-136 are provided in Table 1.

[0137] In some embodiments, the VL comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 138. For example, the VL comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 138.

[0138] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof; and VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136.

[0139] In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 137, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 138.

[0140] The VH the VL can be linked one after another, e.g., by a linker. In some embodiments, the VH and VL are linked via a linker. In some embodiments, the linker 36FH13240754.1Attorney Docket No.: ABH-01225comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO : 4. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. The variable regions from the N- to the C-terminus can be VH-VL or VL-VH.

[0141] In some embodiments, the VH is positioned at the N-terminus of the extracellular domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VH-VL. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the VH and VL are linked via a linker comprising or consisti ng of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139. SEQ ID NO: 139 is provided in Table 1.

[0142] In some embodiments, the VL is positioned at the N-terminus of the extracellular antigen-binding domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VL-VH. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 140. SEQ ID NO: 140 is provided in Table 1.

[0143] In some embodiments, the CDRs are identified according to the Kabat numbering system.37FH13240754.1Attorney Docket No.: ABH-01225Table 1. Amino Acid Sequences of Exemplified Murine Binding Agent GPC3-8

[0144] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 is set forth in SEQ ID NO: 141, which is provided below. CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCAC CTGCACAGTCTCTGGTTTCTCATTAAATAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAG GAAAGGGTCTGGAGTGGCTGGGAGCGATATGGAGTAGTGGAAGCACAGACTATAATGCACCT TTCATATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAACCAAGTTTTCTTTAAAATGAA CAGTCTGCAAGTTGATGACACAGCCATATATTTTTGTGCCGGAAATCCAGAATCGGATCATT ACTACGGCTACGAGGCCATGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO : 141)

[0145] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 is set forth in SEQ ID NO: 142, which is provided below. CAAGTGCAGCTGAAGCAGAGCGGTCCTGGCCTGGTTCAGCCATCTCAGAGCCTGAGCATCAC ATGCACCGTGTCCGGCTTTAGCCTGAACAGCTATGGCGTGCATTGGGTGCGGCAGTCTCCTG GCAAAGGCCTGGAATGGCTGGGCGCCATTTGGTCCAGCGGCTCCACCGACTACAACGCCCCT TTCATCTCTAGACTGAGCATCAGCAAGGACAACAGCAAGAACCAGGTCTTTTTCAAGATGAA CAGCCTGCAAGTGGACGACACCGCCATCTACTTCTGCGCCGGCAACCCCGAGAGCGACCACT38FH13240754.1Attorney Docket No.: ABH-01225ACTACGGCTACGAGGCCATGGACAGCTGGGGCCAGGGCACCAGCGTGACAGTGTCCAGC(SEQ ID NO : 142)

[0146] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 is set forth in SEQ ID NO: 143, which is provided below. GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCAT CACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAGACAGGGAA AATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCCGATGGTGTGCCATCAAGGTTC AGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCATCAGCCTTCAGCCTGAAGATTT TGGGAGTTATTACTGTCAACATTTTTGGACTACTCCATTCACGTTCGGCTCGGGGACAAAGT TGGAAATAAAA (SEQ ID NO : 143)

[0147] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 is set forth in SEQ ID NO: 144, which is provided below. GATATCCAGATGACACAGAGCCCTGCTTCTCTCTCCGCCAGCGTGGGCGAGACAGTGACCAT CACCTGTAGAGCCAGCGGAAATATCCACAACTACCTGGCCTGGTACCAGCAGCGGCAAGGCA AGAGCCCCCAGCTGCTGGTGTACAATGCCAAGACCCTGGCTGATGGCGTGCCCAGCAGATTC AGCGGATCCGGCTCTGGCACACAGTACAGCCTGAAAATCATCAGCCTGCAGCCTGAGGATTT CGGAAGCTACTATTGCCAGCACTTCTGGACCACCCCTTTCACCTTCGGCTCTGGCACTAAGC TGGAAATCAAG (SEQ ID NO : 144)

[0148] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 is set forth in SEQ ID NO: 145, which is provided below. CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCAC CTGCACAGTCTCTGGTTTCTCATTAAATAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAG GAAAGGGTCTGGAGTGGCTGGGAGCGATATGGAGTAGTGGAAGCACAGACTATAATGCACCT TTCATATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAACCAAGTTTTCTTTAAAATGAA CAGTCTGCAAGTTGATGACACAGCCATATATTTTTGTGCCGGAAATCCAGAATCGGATCATT ACTACGGCTACGAGGCCATGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGT GGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCTGACATCCAGATGACTCAGTC TCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGA ATATTCACAATTATTTAGCATGGTATCAGCAGAGACAGGGAAAATCTCCTCAGCTCCTGGTC TATAATGCAAAAACCTTAGCCGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAAC ACAATATTCTCTCAAGATCATCAGCCTTCAGCCTGAAGATTTTGGGAGTTATTACTGTCAAC ATTTTTGGACTACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO : 145)39FH13240754.1Attorney Docket No.: ABH-01225

[0149] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 is set forth in SEQ ID NO: 146, which is provided below. CAAGTGCAGCTGAAGCAGAGCGGTCCTGGCCTGGTTCAGCCATCTCAGAGCCTGAGCATCAC ATGCACCGTGTCCGGCTTTAGCCTGAACAGCTATGGCGTGCATTGGGTGCGGCAGTCTCCTG GCAAAGGCCTGGAATGGCTGGGCGCCATTTGGTCCAGCGGCTCCACCGACTACAACGCCCCT TTCATCTCTAGACTGAGCATCAGCAAGGACAACAGCAAGAACCAGGTCTTTTTCAAGATGAA CAGCCTGCAAGTGGACGACACCGCCATCTACTTCTGCGCCGGCAACCCCGAGAGCGACCACT ACTACGGCTACGAGGCCATGGACAGCTGGGGCCAGGGCACCAGCGTGACAGTGTCCAGCGGC GGAGGCGGATCTGGCGGAGGCGGCTCTGGAGGCGGCGGCTCAGATATCCAGATGACACAGAG CCCTGCTTCTCTCTCCGCCAGCGTGGGCGAGACAGTGACCATCACCTGTAGAGCCAGCGGAA ATATCCACAACTACCTGGCCTGGTACCAGCAGCGGCAAGGCAAGAGCCCCCAGCTGCTGGTG TACAATGCCAAGACCCTGGCTGATGGCGTGCCCAGCAGATTCAGCGGATCCGGCTCTGGCAC ACAGTACAGCCTGAAAATCATCAGCCTGCAGCCTGAGGATTTCGGAAGCTACTATTGCCAGC ACTTCTGGACCACCCCTTTCACCTTCGGCTCTGGCACTAAGCTGGAAATCAAG (SEQ ID NO : 146)

[0150] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 is set forth in SEQ ID NO: 147, which is provided below. GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCAT CACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAGACAGGGAA AATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCCGATGGTGTGCCATCAAGGTTC AGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCATCAGCCTTCAGCCTGAAGATTT TGGGAGTTATTACTGTCAACATTTTTGGACTACTCCATTCACGTTCGGCTCGGGGACAAAGT TGGAAATAAAAGGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCTCAGGTG CAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCAC AGTCTCTGGTTTCTCATTAAATAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGG GTCTGGAGTGGCTGGGAGCGATATGGAGTAGTGGAAGCACAGACTATAATGCACCTTTCATA TCCAGACTGAGCATCAGCAAGGACAACTCCAAGAACCAAGTTTTCTTTAAAATGAACAGTCT GCAAGTTGATGACACAGCCATATATTTTTGTGCCGGAAATCCAGAATCGGATCATTACTACG GCTACGAGGCCATGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO : 147 )

[0151] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 is set forth in SEQ ID NO: 148, which is provided below.40FH13240754.1Attorney Docket No.: ABH-01225GATATCCAGATGACACAGAGCCCTGCTTCTCTCTCCGCCAGCGTGGGCGAGACAGTGACCAT CACCTGTAGAGCCAGCGGAAATATCCACAACTACCTGGCCTGGTACCAGCAGCGGCAAGGCA AGAGCCCCCAGCTGCTGGTGTACAATGCCAAGACCCTGGCTGATGGCGTGCCCAGCAGATTC AGCGGATCCGGCTCTGGCACACAGTACAGCCTGAAAATCATCAGCCTGCAGCCTGAGGATTT CGGAAGCTACTATTGCCAGCACTTCTGGACCACCCCTTTCACCTTCGGCTCTGGCACTAAGC TGGAAATCAAGGGCGGAGGCGGATCTGGCGGAGGCGGCTCTGGAGGCGGCGGCTCACAAGTG CAGCTGAAGCAGAGCGGTCCTGGCCTGGTTCAGCCATCTCAGAGCCTGAGCATCACATGCAC CGTGTCCGGCTTTAGCCTGAACAGCTATGGCGTGCATTGGGTGCGGCAGTCTCCTGGCAAAG GCCTGGAATGGCTGGGCGCCATTTGGTCCAGCGGCTCCACCGACTACAACGCCCCTTTCATC TCTAGACTGAGCATCAGCAAGGACAACAGCAAGAACCAGGTCTTTTTCAAGATGAACAGCCT GCAAGTGGACGACACCGCCATCTACTTCTGCGCCGGCAACCCCGAGAGCGACCACTACTACG GCTACGAGGCCATGGACAGCTGGGGCCAGGGCACCAGCGTGACAGTGTCCAGC (SEQ ID NO : 148 )

[0152] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 360. SEQ ID NO: 359 and SEQ ID NO: 360 are disclosed in Table 2.

[0153] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NOs: 132-133 and 358 are provided in Table 2.

[0154] In some embodiments, the VH comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 359. For example, the VH comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,41FH13240754.1Attorney Docket No.: ABH-01225about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359.

[0155] In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. SEQ ID NOs: 134-136 are provided in Table 2.

[0156] In some embodiments, the VL comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 360. For example, the VL comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 360. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 360.

[0157] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof; and VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid 42FH13240754.1Attorney Docket No.: ABH-01225sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136.

[0158] In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 360.

[0159] The VH the VL can be linked one after another, e.g., by a linker. In some embodiments, the VH and VL are linked via a linker. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO : 4. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. The variable regions from the N- to the C-terminus can be VH-VL or VL-VH.

[0160] In some embodiments, the VH is positioned at the N-terminus of the extracellular domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VH-VL. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 360. In some embodiments, the VH and VL are linked via a linker comprising or consisti ng of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 363. SEQ ID NO: 363 is provided in Table 2.

[0161] In some embodiments, the VL is positioned at the N-terminus of the extracellular antigen-binding domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VL-VH. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the 43FH13240754.1Attorney Docket No.: ABH-01225scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 360. some embodiments

[0162] In some embodiments, the CDRs are identified according to the Kabat numbering system.Table.2 Amino Acid Sequences of Exemplified Humanized Binding Agent GPC3-8-hl

[0163] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 359 is set forth in SEQ ID NO: 373, which is provided below. CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO : 373)

[0164] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 360 is set forth in SEQ ID NO: 374, which is provided below. GACATTCAGATGACCCAAAGCCCCTCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCAT CACCTGTCGCGCGTCTGGCAACATCCACAATTACCTGGCTTGGTACCAGCAGAAACCGGGCA AGGCCCCTAAGCTGCTGGTCTATAACGCCAAGACCCTGGCCGACGGCGTGCCCTCCCGGTTC44FH13240754.1Attorney Docket No.: ABH-01225AGCGGCAGTGGTTCGGGGACTGATTTTACACTGACAATCAGCAGCCTGCAGCCTGAGGACTT CGCCACCTACTACTGCCAGCACTTCTGGACCACCCCATTCACCTTCGGCGGAGGGACCAAGC TCGAGATCAAG (SEQ ID NO : 374)

[0165] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 363 is set forth in SEQ ID NO: 377, which is provided below. CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCggt ggaggtggatcaggtggaggtggatctggtggaggtggatctGACATTCAGATGACCCAAAG CCCCTCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGCGCGTCTGGCA ACATCCACAATTACCTGGCTTGGTACCAGCAGAAACCGGGCAAGGCCCCTAAGCTGCTGGTC TATAACGCCAAGACCCTGGCCGACGGCGTGCCCTCCCGGTTCAGCGGCAGTGGTTCGGGGAC TGATTTTACACTGACAATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGC ACTTCTGGACCACCCCATTCACCTTCGGCGGAGGGACCAAGCTCGAGATCAAG (SEQ ID NO : 377 )

[0166] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 361. SEQ ID NO: 359 and SEQ ID NO: 361 are disclosed in Table 3.

[0167] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NOs: 132-133 and 358 are provided in Table 3.45FH13240754.1Attorney Docket No.: ABH-01225

[0168] In some embodiments, the VH comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 359. For example, the VH comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359.

[0169] In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. SEQ ID NOs: 134, 135, and 136 are provided in Table 3.

[0170] In some embodiments, the VL comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 361. For example, the VL comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 361.

[0171] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof; and VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a 46FH13240754.1Attorney Docket No.: ABH-01225CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136.

[0172] In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 361.

[0173] The VH the VL can be linked one after another, e.g., by a linker. In some embodiments, the VH and VL are linked via a linker. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO : 4. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. The variable regions from the N- to the C-terminus can be VH-VL or VL-VH.

[0174] In some embodiments, the VH is positioned at the N-terminus of the extracellular domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VH-VL. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the VH and VL are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 364. SEQ ID NO: 364 is provided in Table 3.

[0175] In some embodiments, the VL is positioned at the N-terminus of the extracellular antigen-binding domain, i.e., the VH and the VL are positioned from the N- to the C-terminus 47FH13240754.1Attorney Docket No.: ABH-01225as VL-VH. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 361.

[0176] In some embodiments, the CDRs are identified according to the Kabat numbering system.Table.3 Amino Acid Sequences of Exemplified Humanized Binding Agent GPC3-8-h2

[0177] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 359 is set forth in SEQ ID NO: 373, which is provided below. CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT48FH13240754.1Attorney Docket No.: ABH-01225ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO : 373)

[0178] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 361 is set forth in SEQ ID NO: 375, which is provided below. GACATTCAGATGACGCAGTCCCCCTCCAGCCTGAGCGCGTCGGTCGGCGACCGCGTGACCAT CACCTGTAGAGCCAGCGGCAACATCCACAATTACCTGGCCTGGTACCAGCAGAAGCCAGGCA AGGCCCCTAAACTGCTGGTGTACAACGCCAAGACCCTGGCTGATGGCGTGCCCTCCCGGTTC AGCGGCAGCGGATCTGGCACCGACTACACACTGACCATCAGCTCTCTGCAGCCTGAGGACTT CGCCACATACTACTGCCAGCACTTCTGGACCACCCCGTTTACCTTCGGCGGAGGTACGAAGC TCGAGATCAAG (SEQ ID NO : 375)

[0179] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 364 is set forth in SEQ ID NO: 378, which is provided below. CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCggt ggaggtggatcaggtggaggtggatctggtggaggtggatctGACATTCAGATGACGCAGTC CCCCTCCAGCCTGAGCGCGTCGGTCGGCGACCGCGTGACCATCACCTGTAGAGCCAGCGGCA ACATCCACAATTACCTGGCCTGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAACTGCTGGTG TACAACGCCAAGACCCTGGCTGATGGCGTGCCCTCCCGGTTCAGCGGCAGCGGATCTGGCAC CGACTACACACTGACCATCAGCTCTCTGCAGCCTGAGGACTTCGCCACATACTACTGCCAGC ACTTCTGGACCACCCCGTTTACCTTCGGCGGAGGTACGAAGCTCGAGATCAAG (SEQ ID NO : 378 )

[0180] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 362. SEQ ID NO: 359 and SEQ ID NO: 362 are disclosed in Table 4.

[0181] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative49FH13240754.1Attorney Docket No.: ABH-01225modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NOs: 132-133 and 358 are provided in Table 4.

[0182] In some embodiments, the VH comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 183. For example, the VH comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359.

[0183] In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. SEQ ID NOs: 134-136 are provided in Table 4.

[0184] In some embodiments, the VL comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 362. For example, the VL comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 362.50FH13240754.1Attorney Docket No.: ABH-01225

[0185] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof; and VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136.

[0186] In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 359, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 362.

[0187] The VH the VL can be linked one after another, e.g., by a linker. In some embodiments, the VH and VL are linked via a linker. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO : 4. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. The variable regions from the N- to the C-terminus can be VH-VL or VL-VH.

[0188] In some embodiments, the VH is positioned at the N-terminus of the extracellular domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VH-VL. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL 51FH13240754.1Attorney Docket No.: ABH-01225comprising the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the VH and VL are linked via a linker comprising or consisti ng of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 365. SEQ ID NO: 365 is provided in Table 4.

[0189] In some embodiments, the VL is positioned at the N-terminus of the extracellular antigen-binding domain, i.e., the VH and the VL are positioned from the N- to the C-terminus as VL-VH. In some embodiments, the extracellular domain of the CAR is an scFv that comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 162.

[0190] In some embodiments, the CDRs are identified according to the Kabat numbering system.Table.4 Amino Acid Sequences of Exemplified Humanized Binding Agent GPC3-8-h3

[0191] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 359 is set forth in SEQ ID NO: 373, which is provided below.52FH13240754.1Attorney Docket No.: ABH-01225CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO : 373)

[0192] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 362 is set forth in SEQ ID NO: 376, which is provided below. GACATCCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCAT CACCTGTCGCGCAAGTGGCAACATCCACAACTACCTGGCCTGGTACCAGCAGAAACCCGGCA AGGCGCCACAGCTGCTGGTCTACAACGCCAAGACCCTGGCTGATGGCGTGCCCTCTCGGTTT TCGGGCAGCGGATCTGGCACACAGTATACATTGACCATCAGCAGCCTCCAGCCTGAGGACTT CGCCACCTACTACTGCCAGCACTTCTGGACCACCCCTTTCACCTTCGGCGGAGGCACTAAGC TGGAAATTAAG (SEQ ID NO : 376)

[0193] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 365 is set forth in SEQ ID NO: 379, which is provided below. CAGGTGCAGCTCCAAGAGTCCGGACCGGGTCTTGTCAAACCCAGCGAGACACTGTCTCTGAC CTGTACCGTCTCTGGCTTCAGCCTGAACAGCTATGGCGTGCACTGGATCCGCCAGCCTCCAG GCAAGGGCCTGGAATGGCTGGGCGCCATCTGGTCCAGCGGCAGCACCGACTACAACGCCCCT TTCATCAGCAGACTGACTATTAGCAAGGATACCTCCAAGAACCAGGTTTTTCTGAAGCTGAG CAGCGTGACAGCTGCTGATACCGCCGTGTACTTCTGCGCCGGCAATCCCGAGAGCGACCACT ACTACGGCTACGAGGCGATGGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCggt ggaggtggatcaggtggaggtggatctggtggaggtggatctGACATCCAGATGACCCAGTC CCCCTCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGCGCAAGTGGCA ACATCCACAACTACCTGGCCTGGTACCAGCAGAAACCCGGCAAGGCGCCACAGCTGCTGGTC TACAACGCCAAGACCCTGGCTGATGGCGTGCCCTCTCGGTTTTCGGGCAGCGGATCTGGCAC ACAGTATACATTGACCATCAGCAGCCTCCAGCCTGAGGACTTCGCCACCTACTACTGCCAGC ACTTCTGGACCACCCCTTTCACCTTCGGCGGAGGCACTAAGCTGGAAATTAAG (SEQ ID NO : 379)

[0194] The VH and / or VL amino acid sequences comprising or consisting of at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g. , about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 53FH13240754.1Attorney Docket No.: ABH-0122588%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a specific sequence (e.g., SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 362) may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the specified sequence(s), but retain the ability to bind to a target antigen (e.g., GPC3). In some embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in a specific sequence (e.g., SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 362). In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) of the extracellular antigen-binding domain. In some embodiments, the extracellular domain comprises VH and / or VL sequence selected from SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 362, including post-translational modifications of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 362.

[0195] In addition, the extracellular domain can comprise a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum. Signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway. In some embodiments, the signal peptide is covalently joined to the 5’ terminus (N-terminus) of the extracellular domain. In some embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In some embodiments, the signal peptide is generated from the antibody from it is derived. In some embodiments, the signal peptide is linked to the N-terminus of a VH disclosed herein. In some embodiments, the signal peptide is linked to the N-terminus of a VL disclosed herein. In some embodiments, the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, or SEQ ID NO: 289.MGWSCI IFFLVATATGVLS (SEQ ID NO: 274 )MKCSWVIFFLMAWTGVNS (SEQ ID NO: 275 )MNFGLRLIFLVLVLKGVLC (SEQ ID NO: 276)FH13240754.1Attorney Docket No.: ABH-01225MGWSCI ILFLVATATGVHS (SEQ ID NO: 277 )MGWSCI IFFLVATATGVHS (SEQ ID NO: 278 )MGWSSI ILFLVATASGVHS (SEQ ID NO: 279)MAVLGLLFCLVTFP SCVLS (SEQ ID NO: 280 )MSPAQFLFLSVLWIRETNG (SEQ ID NO: 281 )MSPAQFLFLLVLWIREIHG (SEQ ID NO: 282 )MDSQAQVLMLLLLWVSGTCG (SEQ ID NO : 283)MSPAQFLFLLVLWNRETNG (SEQ ID NO: 284 )MESHTQAFVFAFLWLSGVDG (SEQ ID NO : 285)MSPAQFLFLLVLWIRETNG (SEQ ID NO: 286)MRVPAHVFGFLLLWFPGTRC (SEQ ID NO : 287)MSVLTQVLALLLLWLTGARC (SEQ ID NO : 288)MRVLAELLGLLLFCFLGVRC (SEQ ID NO : 289)

[0196] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 274 is set forth in SEQ ID NO: 290. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 275 is set forth in SEQ ID NO: 291. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 276 is set forth in SEQ ID NO: 292. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 277 is set forth in SEQ ID NO: 293 or SEQ ID NO: 294. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 278 is set forth in SEQ ID NO: 295. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 279 is set forth in SEQ ID NO: 296. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 280 is set forth in SEQ ID NO: 297. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 281 is set forth in SEQ ID NO: 298. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 282 is set forth in SEQ ID NO: 299. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 283 is set forth in SEQ ID NO: 300. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 284 is set forth in SEQ ID NO: 301. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 285 is set forth in SEQ ID NO: 302. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 286 is set forth in SEQ ID NO: 303 or SEQ ID NO: 304. In some embodiments, an 55FH13240754.1Attorney Docket No.: ABH-01225exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 287 is set forth in SEQ ID NO: 305. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 288 is set forth in SEQ ID NO: 306. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 289 is set forth in SEQ ID NO: 307. SEQ ID NOs: 290-307 are provided below.ATGGGTTGGAGCTGTATCATCTTCTTTCTGGTAGCAACAGCTACAGGTGTGCTCTCC ( SEQ ID NO : 290) ATGAAATGCAGCTGGGTTATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCA (SEQ ID NO : 291) ATGAACTTCGGGCTCAGATTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCTGTGT (SEQ ID NO : 292 )ATGGGTTGGAGCTGTATCATCCTTTTTCTGGTAGCAACAGCTACAGGTGTGCACTCC (SEQ ID NO: 293)ATGGGCTGGTCATGCATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATAGT (SEQ ID NO: 294 )ATGGGTTGGAGCTGTATCATCTTCTTTCTGGTAGCAACAGCTACAGGTGTGCACTCC (SEQ ID NO: 295)ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCCTCAGGTGTCCACTCC (SEQ ID NO: 296)ATGGCTGTCTTGGGGCTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTATCC (SEQ ID NO: 297 )ATGAGTCCTGCCCAGTTCCTGTTTCTGTCAGTGCTCTGGATTCGGGAAACCAACGGT (SEQ ID NO: 298)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAATCCACGGT (SEQ ID NO: 299)ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGG (SEQ ID NO : 300)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGAATCGGGAAACCAACGGT (SEQ ID NO: 301)ATGGAGTCTCATACTCAGGCCTTTGTATTCGCGTTTCTCTGGTTGTCTGGTGTTGATGGA (SEQ ID NO : 302)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAATGGT (SEQ ID NO: 303)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGT (SEQ ID NO: 304 )56FH13240754.1Attorney Docket No.: ABH-01225 ATGAGGGTTCCTGCTCACGTTTTTGGCTTCTTGTTGCTCTGGTTTCCAGGTACCAGATGT (SEQ ID NO : 305) ATGAGTGTGCTCACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT (SEQ ID NO : 306) ATGAGGGTCCTTGCTGAGCTCCTGGGGCTGCTGCTGTTCTGCTTTTTAGGTGTGAGATGT (SEQ ID NO : 307)

[0197] In some embodiments, the extracellular domain of the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VH.

[0198] In some embodiments, the extracellular domain of the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VH.

[0199] In some embodiments, the extracellular domain of the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 138, and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VL.

[0200] In some embodiments, the extracellular domain of the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 360 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VL.

[0201] In some embodiments, the extracellular domain of the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 361 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VL.

[0202] In some embodiments, the extracellular domain of the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 362 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the VL.

[0203] In some embodiments, the extracellular domain of the CAR comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 139, and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the scFv.57FH13240754.1Attorney Docket No.: ABH-01225

[0204] In some embodiments, the extracellular domain of the CAR comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 140 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the scFv.

[0205] In some embodiments, the extracellular domain of the CAR comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 363 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the scFv.

[0206] In some embodiments, the extracellular domain of the CAR comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 364 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the scFv.

[0207] In some embodiments, the extracellular domain of the CAR comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 365 and a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 280. In some embodiments, the signal peptide is linked to the N-terminus of the scFv.Transmembrane Domain of a CAR

[0208] In some embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal are transmitted to the cell.

[0209] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (e.g., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD27, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, CD154, ICAM-1, KIRDS2, 0X40, CD2, NKGD2, LFA-1 (CDlla, CD18) , ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1), CD160, CD166, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRT AM, Ly958FH13240754.1Attorney Docket No.: ABH-01225(CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8) , SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, and PAG / Cbp. In some embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of CD8, of CD28, of CD3^, of CD4, of 4-1BB, of 0X40, of ICOS, of CD84, of CD166, of CD8a, of CD8b, of ICAM-1, of CTLA-4, of CD27, of CD40, of NKGD2, or a combi nation thereof. Alternati vely, the transmembrane domain may be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular domain of the CAR.

[0210] In some embodiments, the CAR comprises more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.

[0211] In some embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD28 or a portion thereof. The CD28 polypeptide can comprise or consist of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical or homologous to the sequence having a NCBI Reference No: NP_006130 (SEQ ID NO: 308), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In some embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 308, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, and / or up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, or up to about 220 amino acids in length. In some embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 154 to 179, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 308. In some embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 154 to 179 of SEQ ID NO: 308. SEQ ID NO: 308 is provided below.59FH13240754.1Attorney Docket No.: ABH-01225MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSA VEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDI YFCKIEVMYPPP YLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO : 308 ]

[0212] An exemplary nucleotide sequence encoding the amino acid sequence of amino acids 154 to 179 of SEQ ID NO: 308 is set forth in SEQ ID NO: 309, which is provided below.TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTG [SEQ ID NO : 309 ]

[0213] In some embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., a transmembrane domain of CD8 or a portion thereof). In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD8 or a portion thereof. In some embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the sequence having a NCBI Reference No: NP_001139345.1 (SEQ ID NO: 310) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservati ve amino acid substitutions. In some embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 310, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 235 amino acids in length. Alternatively or additionally, in some embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 137 to 209 or 200 to 235 of SEQ ID NO: 310. In some embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of the amino acid sequence of amino acids 137 to 209 of SEQ ID NO: 310. SEQ ID NO: 310 is provided below.MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS IMY FSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [SEQ ID NO : 310 ]

[0214] In some embodiments, the CAR further comprise a spacer / hinge domain that links the extracellular domain to the transmembrane domain. A hinge domain is a short sequence 60FH13240754.1Attorney Docket No.: ABH-01225of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol.4(2): 89-99 (2004)). The hinge domain can be any suitable sequence derived or obtained from any suitable molecule. The hinge domain can be flexible enough to allow the extracellular domain to orient in different directions to facilitate antigen recognition while preserving the activating activity of the CAR.

[0215] In some embodiments, for example, the hinge domain sequence is derived from a CD8a molecule or a CD28 molecule.

[0216] In some embodiments, the hinge / spacer domain of the CAR comprises a nati ve or modified hinge region of CD8, of CD28, of CD3^, of CD40, of 4-1BB, of 0X40, of CD84, of CD 166, of CD8a, of CD8b, of ICOS, of ICAM-1, of CTLA-4, of CD27, of CD40, of NKGD2, or a combination thereof. The hinge / spacer region can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide (I., a portion of SEQ ID NO: 308), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 310), a variant of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identical or homologous thereto, or a synthetic spacer sequence.

[0217] In some embodiments, the hinge domain of the CAR comprises a native or modified hinge region of CD28. In some embodiments, the hinge domain of the CAR comprises a native hinge region of CD28. In some embodiments, the hinge domain of the CAR comprises the amino acid sequence of amino acids 114 to 153 of SEQ ID NO: 308. An exemplary nucleotide sequence encoding the amino acid sequence of amino acids 114 to 153 of SEQ ID NO: 308 is set forth in SEQ ID NO: 311, which is provided below.ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCA TGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTT[SEQ ID NO : 311]Intracellular Domain of a CAR

[0218] In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the intracellular domain of the CAR comprises a CD3^ polypeptide. CD3^ can activate or stimulate a cell (e.g., an immune effector cell, e.g., a cell of the lymphoid lineage, e.g., a T-cell) after antigen recognition. The intracellular domain of the CD3^-chain is the primary transmitter of signals from endogenous TCRs.

[0219] In some embodiments, the intracellular domain of the CAR transmits a signal to the immune effector cell expressing the CAR after antigen recognition, activating at least one 61FH13240754.1Attorney Docket No.: ABH-01225of the normal effector functions of the immune effector cell. In some embodiments, the effector function of a T cell, for example, is cytolytic activity or helper activity, including secretion of cytokines. Therefore, the intracellular domain can comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co-receptors. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.

[0220] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs). Examples of IT AM containing cytoplasmic signaling sequences include those derived from CD8, CD3^, CD35, CD3y, CD3a, CD32 (Fc gamma Rlla), DAP10, DAP12, CD79a, CD79b, FcyRIy, FcyRIIIy, FcaRip (FCERIB), and FcaRIy (FCERIG).

[0221] In some embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3Q (TCR zeta, GenBank acc. no. BAG36664.1). T-cell surface glycoprotein CD3 zeta (CD3Q chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene. Wild type (“native”) CD3^ comprises three functional immunoreceptor tyrosine-based activation motifs (IT AMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3). The intracellular tails of the CD3 molecules comprise a single ITAM, which is responsible for the signaling capacity of the TCR. The intracellular tail of the C, chain (CD3Q comprises three (3) IT AMs.

[0222] In some embodiments, the intracellular domain of the CAR comprises a native CD3^. In some embodiments, the native CD3^ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence having a NCBI Reference No: NP_932170 (SEQ ID NO: 312), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In some embodiments, the CD3^ polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 312, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In some embodiments, the native CD3^ comprises or consists of the amino 62FH13240754.1Attorney Docket No.: ABH-01225acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 312. In some embodiments, the intracellular signaling domain of the CAR comprises a native CD3^ comprising or consisting of the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 312. SEQ ID NO: 312 is provided below.MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO : 312 ]

[0223] In some embodiments, the intracellular domain of the CAR comprises a modified CD3^ polypeptide. In some embodiments, the modified CD3^ polypeptide comprises a mutation, such as a point mutation, in at least one IT AM so as to render the IT AM nonfunctional. In some embodiments, either the membrane-proximal IT AM (IT AMI), the membrane-distal IT AM (C-terminal third IT AM, ITAM3), or both are non-functional. In some embodiments, either two membrane-proximal IT AMs (IT AMI and ITAM2) or two membrane-distal IT AMs (ITAM2 and ITAM3) are non-functional. In some embodiments, only ITAM2 is non-functional. In some embodiments, the modified CD3^ polypeptide comprises a deletion (e.g., a truncation) mutation such that at least one IT AM is missing. In some embodiments, the modified CD3^ polypeptide is missing the membrane-proximal IT AM (IT AMI), the membrane-distal IT AM (ITAM3), or both. In other embodiments, the modified CD3^ polypeptide is missing either two membrane-proximal ITAMs (IT AMI and ITAM2) or two membrane-distal ITAMs (ITAM2 and ITAM3). In some embodiments, the modified CD3^ polypeptide is missing ITAM2. Removing at least one IT AM from the introduced CAR may reduce CD3^-mediated apoptosis. Alternatively, removing at least one IT AM from the introduced CAR can reduce its size without loss of function.

[0224] In some embodiments, the modified CD3^ polypeptide comprises one, two or three ITAMs. In some embodiments, the modified CD3^ polypeptide comprises a native IT AMI. In some embodiments, the native IT AMI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 313.QNQLYNELNLGRREEYDVLDKR [SEQ ID NO : 313]

[0225] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 313 is set forth in SEQ ID NO: 314, which is provided below.CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA GAGA [SEQ ID NO : 314 ]63FH13240754.1Attorney Docket No.: ABH-01225

[0226] In some embodiments, the modified CD3^ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In some embodiments, the IT AMI variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss of function mutations comprises a mutation of a tyrosine residue in IT AMI. In some embodiments, the ITAM1 variant consists of two loss-of-function mutations. In some embodiments, the IT AMI variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 315, which is provided below. QNQLFNELNLGRREEFDVLDKR [SEQ ID NO : 315]

[0227] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 315 is set forth in SEQ ID NO: 316, which is provided below.CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAA GAGA [SEQ ID NO : 316]

[0228] In some embodiments, the modified CD3^ polypeptide comprises a native ITAM2. In some embodiments, the native ITAM2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 317, which is provided below.QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO : 317 ]

[0229] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 317 is set forth in SEQ ID NO: 318, which is provided below.CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAA [SEQ ID NO : 318 ]

[0230] In some embodiments, the modified CD3^ polypeptide comprises an ITAM2 variant. In some embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM2. In some embodiments, the IT AMI variant consists of two loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 319, which is provided below.QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO : 319 ]

[0231] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 319 is set forth in SEQ ID NO: 320, which is provided below.CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGG GATGAAA [SEQ ID NO : 320 ]64FH13240754.1Attorney Docket No.: ABH-01225

[0232] In some embodiments, the modified CD3^ polypeptide comprises a native ITAM3. In some embodiments, the native ITAM3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 321, which is provided below.HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO : 321]

[0233] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 321 is set forth in SEQ ID NO: 322, which is provided below.CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAG [SEQ ID NO : 322 ]

[0234] In some embodiments, the modified CD3^ polypeptide comprises an ITAM3 variant. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM3. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 323, which is provided below.HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO : 323]

[0235] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 323 is set forth in SEQ ID NO: 324, which is provided below.CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACAT GCAG [SEQ ID NO : 324 ]

[0236] Various modified CD3^ polypeptides and CARs comprising modified CD3^ polypeptides are disclosed in International Patent Application Publication No.WO2019 / 133969, which is incorporated by reference hereby in its entirety.

[0237] In some embodiments, the intracellular domain of the CAR comprises a modified CD3^ polypeptide comprising a native IT AMI, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more e.g., two) loss-of-function mutations. In some embodiments, the intracellular domain of the CAR comprises a modified CD3^ polypeptide comprising a native IT AMI, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In some embodiments, the intracellular domain of the CAR comprises a modified CD3^ polypeptide comprising a native IT AMI comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 313, an ITAM2 variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 319,65FH13240754.1Attorney Docket No.: ABH-01225and an ITAM3 variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 323. In some embodiments, the modified CD3^ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 325. SEQ ID NO: 325 is provided below. In some embodiments, a CAR comprising an intracellular domain comprising a modified CD3^ polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 325 is designated as an “1XX” CAR.RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNEL QKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO : 325]

[0238] In some embodiments, the intracellular domain of the CAR comprises a modified CD3^ polypeptide comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO: 325 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0239] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 325 is set forth in SEQ ID NO: 326, which is provided below.AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO : 326 ]

[0240] In some embodiments, the intracellular domain of the CAR further comprises at least one co- stimulatory signaling region. In some embodiments, the at least one costimulatory region comprises a co-stimulatory molecule or a portion thereof. In some embodiments, the at least one co-stimulatory region comprises an intracellular domain of at least one co-stimulatory molecule or a portion thereof.

[0241] As used herein, a “co-stimulatory molecule” refers to a cell surface molecule other than antigen receptor or its ligand that can provide an efficient response of lymphocytes to an antigen. A co-stimulatory molecule can provide optimal lymphocyte activation. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on a cell surface that upon binding to its receptor produces a co-stimulatory response, i.e., an 66FH13240754.1Attorney Docket No.: ABH-01225intracellular response that effects the stimulation provided when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen. As one example, a4-lBB ligand (z.e., 4-1BBL) may bind to 4-1BB for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR+T-cell.

[0242] Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB (CD137), 0X40, ICOS, DAP-10, CD27, CD28, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD8, CD4, B2C, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. and combinations thereof.

[0243] In some embodiments, the intracellular domain of the CAR comprises a costimulatory signaling region that comprises a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In some embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of human CD28 or a portion thereof.

[0244] In some embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the CAR comprise or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 308, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservati ve amino acid substitutions. In some embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region comprises or consist of an amino acid sequence that is a consecutive portion of SEQ ID NO: 308, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. Alternatively or additionally, in some embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of SEQ ID NO: 308. In some embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 308.67FH13240754.1Attorney Docket No.: ABH-01225

[0245] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 308 is set forth in SEQ ID NO: 327, which is provided below.AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGG GCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC[SEQ ID NO : 327 ]

[0246] In some embodiments, the intracellular domain of the CAR comprises a modified CD28 domain that imparts unique functional properties to the CAR. In this regard, a native CD28 domain comprises three intracellular subdomains consisting of the amino acid sequences YMNM (SEQ ID NO: 383), PRRP (SEQ ID NO: 384), and PYAP (SEQ ID NO: 385) that regulate signaling pathways post stimulation. In some embodiments, the intracellular domain of the CAR comprises a modified CD28 domain wherein one or more of the YMNM (SEQ ID NO: 383), PRRP (SEQ ID NO: 384), and PYAP (SEQ ID NO: 385) subdomains are mutated or deleted, so as to amplify, attenuate, or inactivate the subdomain(s), thereby modulating CAR-T function. In some embodiments, the intracellular domain of the CAR comprises a modified CD28 domain disclosed in International Patent Application Publication Nos.: WO 2019 / 010383 and WO 2021158850, which are incorporated by reference in their entireties.

[0247] In some embodiments, the intracellular domain of the CAR comprises a costimulatory signaling region that comprises a4-lBB polypeptide, e.g., an intracellular domain of 4- IBB or a portion thereof. In some embodiments, the co-stimulatory signaling region comprises an intracellular domain of human 4- IBB or a portion thereof. In some embodiments, the 4-1BB comprised in the co-stimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to the sequence having a NCBI Ref No.: NP_001552 (SEQ ID NO: 328) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In some embodiments, the 4-1BB comprised in the co-stimulatory signaling region comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 328, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, or up to about 255 amino acids in length. In some embodiments, the 4- IBB 68FH13240754.1Attorney Docket No.: ABH-01225polypeptide comprised in the co-stimulatory signaling region comprises or consists of the amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255 of SEQ ID NO: 328. In some embodiments, the co-stimulatory signaling region comprises a 4- IBB polypeptide comprising or consisting of the amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 328. SEQ ID NO: 328 is provided below:MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTC DICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGT FNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ IISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCEL [SEQ ID NO : 328 ]

[0248] In some embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises intracellular domains of two or more co-stimulatory molecules or portions thereof, e.g., an intracellular domain of CD28 or a portion thereof and an intracellular domain of 4- IBB or a portion thereof, or an intracellular domains of CD28 or a portion thereof and an intracellular domain of 0X40 or a portion thereof.

[0249] In some embodiments, the intracellular domain of the CAR further comprises a peptide. In some embodiments, the peptide is a self-cleaving P2A peptide. In some embodiments, the self-cleaving P2A peptide comprises or consists of the amino acid sequence of SEQ ID NO: 329. SEQ ID NO: 329 is provided below.GSGATNFSLLKQAGDVEENPGP [SEQ ID NO : 329]

[0250] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 329 is set forth in SEQ ID NO: 330, which is provided below.GGATCTGGAGCAACAAACTTCTCACTACTCAAACAAGCAGGGGACGTGGAGGAGAATCCCGG ACCC [SEQ ID NO : 330 ]

[0251] In some embodiments, the CAR is expressed with a C-terminal LNGFR tag. In some embodiments, the LNGFR tag comprises or consists of the amino acid sequence of SEQ ID NO: 331. SEQ ID NO: 331 is provided below.MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGRSSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPWT RGTTDNLIPVYCSILAAVWGLVAYIAFKRWNS [SEQ ID NO : 331 ]69FH13240754.1Attorney Docket No.: ABH-01225

[0252] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 331 is set forth in SEQ ID NO: 332, which is provided below.ATGGGGGCAGGGGCCACCGGACGCGCAATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCT GGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTG AGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTG CAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACG ACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCG TGCCGCGTGTGCGAGGCGGGCTCGGGCCGCTCGAGCGGCCTCGTGTTCTCCTGCCAGGACAA GCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGG ACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGC TGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGA GGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCA TAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACC CGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGG CCTTGTGGCCTACATAGCCTTCAAGAGGTGGAACAGCTGA [SEQ ID NO : 332 ]

[0253] In some embodiments, the CAR is expressed with a C-terminal TGF dominant negative receptor (DNR). In some embodiments, the TGF -DNR modulates, interferes with, or inhibits the inhibitory TGFp signaling to a cell comprising the CAR. In some embodiments, the TGF -DNR comprises or consists of the amino acid sequence of SEQ ID NO: 366. SEQ ID NO: 366 is provided below.MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIF YCYRVNRQQK [SEQ ID NO : 366]

[0254] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 366 is set forth in SEQ ID NO: 381, which is provided below.ATGGGTAGAGGTTTGCTCAGGGGTCTCTGGCCTTTGCACATTGTACTTTGGACGCGAATCGC TTCCACCATCCCCCCTCACGTTCAAAAGAGCGTGAACAATGATATGATTGTAACAGATAATA ACGGGGCTGTCAAATTTCCGCAACTCTGTAAATTCTGCGATGTTCGGTTTAGTACCTGCGAT AACCAGAAATCCTGCATGTCTAATTGTTCTATCACCTCCATCTGTGAAAAACCTCAAGAGGT TTGCGTGGCTGTTTGGAGAAAGAATGACGAAAACATTACGCTTGAGACTGTGTGCCACGACC CCAAACTCCCTTACCACGATTTTATACTGGAGGACGCCGCCAGCCCCAAATGTATTATGAAG70FH13240754.1Attorney Docket No.: ABH-01225GAGAAGAAGAAGCCTGGAGAGACTTTCTTTATGTGTAGTTGTTCTAGTGATGAGTGTAACGA TAATATTATTTTCTCTGAAGAATACAACACTAGCAATCCCGATCTTCTTCTCGTAATATTTC AGGTTACAGGGATTTCCCTCTTGCCTCCACTCGGGGTAGCAATCTCAGTGATAATCATTTTC TATTGCTACCGCGTAAATCGCCAACAAAAA [SEQ ID NO : 381]

[0255] In some embodiments, the CAR comprises a CD28 transmembrane and intracellular co-stimulation domain. In some embodiments, the CD28 transmembrane and intracellular co-stimulation domain can comprise or consist of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical or homologous to the sequence of SEQ ID NO: 367, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservati ve amino acid substitutions. In some embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 367. SEQ ID NO: 367 is provided below.AIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAF IIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO :367 ]

[0256] An exemplified nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 367 is set forth in SEQ ID NO: 380, which is provided below.GCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTAT CCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTT GGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT ATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGAC TCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC [SEQ ID NO : 380 ]Exemplified CARs

[0257] In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 133, and (ii) a VL that comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 135, and a 71FH13240754.1Attorney Docket No.: ABH-01225CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 136, (b) a CD28 transmembrane and intracellular co-stimulation domain comprising or consisti ng of the amino acid sequence set forth in SEQ ID NO: 367, and (c) an intracellular signaling domain comprising a CD3^ polypeptide (e.g., a modified human CD3^ polypeptide, e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 325),. In some embodiments, the CAR further comprises a self-cleaving P2A peptide (e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the CAR further comprises a signal peptide (e.g., one consisting of amino acid sequence set forth in SEQ ID NO: 278). In some embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CAR further comprises a C-terminal TGF dominant negative receptor (DNR), e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 366.

[0258] In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 137, and (ii) a VL that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 138, (b) a CD28 transmembrane and intracellular co-stimulation domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 367, and (c) an intracellular signaling domain comprising a CD3^ polypeptide (e.g., a modified human CD3^ polypeptide, e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 325). In some embodiments, the CAR further comprises a self-cleaving P2A peptide (e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the CAR further comprises a signal peptide (e.g., one consisting of amino acid sequence set forth in SEQ ID NO: 278). In some embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CAR further comprises a C-terminal TGF dominant negative receptor (DNR), e.g., one comprising or consisting of the amino acid sequence of SEQ ID NO: 366. In some embodiments, the VH and VL are positioned from the N- to the C-terminus: VH-VL. In some embodiments, the CAR is designed as “GPC3-8-TGFP-DNR”. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 382, which is provided below.MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLNSYGVHWVRQSPGK GLEWLGAIWSSGSTDYNAPFISRLSISKDNSKNQVFFKMNSLQVDDTAIYFCAGNPESDHYY GYEAMDSWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNI72FH13240754.1Attorney Docket No.: ABH-01225HNYLAWYQQRQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKIISLQPEDFGSYYCQHF WTTPFTFGSGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV LVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLF NELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRGSGATNFSL LKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLL PPLGVAISVIIIFYCYRVNRQQK [SEQ ID NO : 382 ]

[0259] In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 359, and (ii) a VL that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 360, (b) a CD28 transmembrane and intracellular co-stimulation domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 367, and (c) an intracellular signaling domain comprising a CD3^ polypeptide (e.g., a modified human CD3^ polypeptide, e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 325),. In some embodiments, the CAR further comprises a self-cleaving P2A peptide (e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the CAR further comprises a signal peptide (e.g., one consisting of amino acid sequence set forth in SEQ ID NO: 278). In some embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CAR further comprises a C-terminal TGF dominant negative receptor (DNR), e.g., one comprising or consisting of the amino acid sequence of SEQ ID NO: 366.

[0260] In some embodiments, the VH and VL are positioned from the N- to the C-terminus: VH-VL. In some embodiments, the CAR is designed as “GPC3-8-hl-TGFP-DNR”. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 370, which is provided below.MAVLGLLFCLVTFPSCVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLNSYGVHWIRQPPGK GLEWLGAIWSSGSTDYNAPFISRLTISKDTSKNQVFLKLSSVTAADTAVYFCAGNPESDHYY GYEAMDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASGNI HNYLAWYQQKPGKAPKLLVYNAKTLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHF WTTPFTFGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV73FH13240754.1Attorney Docket No.: ABH-01225LVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLF NELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRGSGATNFSL LKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLL PPLGVAISVIIIFYCYRVNRQQK [SEQ ID NO : 370 ]

[0261] In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 359, and (ii) a VL that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 361, (b) a CD28 transmembrane and intracellular co-stimulation domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 367, and (c) an intracellular signaling domain comprising a CD3^ polypeptide (e.g., a modified human CD3^ polypeptide, e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 325),. In some embodiments, the CAR further comprises a self-cleaving P2A peptide (e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the CAR further comprises a signal peptide (e.g., one consisting of amino acid sequence set forth in SEQ ID NO: 278). In some embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CAR further comprises a C-terminal TGF dominant negative receptor (DNR), e.g., one comprising or consisting of the amino acid sequence of SEQ ID NO: 366.

[0262] In some embodiments, the VH and VL are positioned from the N- to the C-terminus: VH-VL. In some embodiments, the CAR is designed as “GPC3-8-h2-TGFP-DNR”. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 368, which is provided below.MAVLGLLFCLVTFPSCVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLNSYGVHWIRQPPGK GLEWLGAIWSSGSTDYNAPFISRLTISKDTSKNQVFLKLSSVTAADTAVYFCAGNPESDHYY GYEAMDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASGNI HNYLAWYQQKPGKAPKLLVYNAKTLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF WTTPFTFGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV LVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLF74FH13240754.1Attorney Docket No.: ABH-01225NELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRGSGATNFSL LKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLL PPLGVAISVIIIFYCYRVNRQQK [SEQ ID NO : 368 ]

[0263] In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 359, and (ii) a VL that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 362, (b) a CD28 transmembrane and intracellular co-stimulation domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 367, and (c) an intracellular signaling domain comprising a CD3^ polypeptide (e.g., a modified human CD3^ polypeptide, e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 325),. In some embodiments, the CAR further comprises a self-cleaving P2A peptide (e.g., one comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 329. In some embodiments, the CAR further comprises a signal peptide (e.g., one consisting of amino acid sequence set forth in SEQ ID NO: 278). In some embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CAR further comprises a C-terminal TGF dominant negative receptor (DNR), e.g., one comprising or consisting of the amino acid sequence of SEQ ID NO: 366.

[0264] In some embodiments, the VH and VL are positioned from the N- to the C-terminus: VH-VL. In some embodiments, the VH- is positioned N-terminal relative to the VL. In some embodiments, the CAR is designed as “GPC3-8-h3-TGFP-DNR”. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 369, which is provided below.MAVLGLLFCLVTFPSCVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLNSYGVHWIRQPPGK GLEWLGAIWSSGSTDYNAPFISRLTISKDTSKNQVFLKLSSVTAADTAVYFCAGNPESDHYY GYEAMDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASGNI HNYLAWYQQKPGKAPQLLVYNAKTLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHF WTTPFTFGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV LVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLF NELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRGSGATNFSL75FH13240754.1Attorney Docket No.: ABH-01225LKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLL PPLGVAISVIIIFYCYRVNRQQK [SEQ ID NO : 369 ]Cells

[0265] Provided herein are cells comprising a GPC3-targeted CAR. In some embodiments, the cell is a cell of lymphoid lineage or a cell of myeloid lineage. In some embodiments, the cell is an immunoresponsive cell. In some embodiments, the immunoresponsive cell is a cell of lymphoid lineage.

[0266] In some embodiments, the cell is a cell of the lymphoid lineage. Cells of the lymphoid lineage can provide production of antibodies, regulation of cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells may be differentiated. In some embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell or an induced pluripotent stem cell).

[0267] In some embodiments, the cell is an immune effector cell. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal el utriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection76FH13240754.1Attorney Docket No.: ABH-01225using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0268] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0269] In some embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0270] The T cells can be any type of T cells, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), Regulatory T cells (also known as suppressor T cells or Treg), tumorinfiltrating lymphocyte (TIL), Natural killer T cells (NK-T cells), mucosal associated invariant T cells, af> T cells, and y5 T cells. In some embodiments, the T cell is a CAR-T cell (i.e., a T cell expressing a CAR).

[0271] In some embodiments, the cell comprising GPC3-targeted CAR is a helper T cell or CD4+T cell. Helper T cells (or T helper cells, or TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH17, TH9, or TEH, which secrete different cytokines to facilitate a different type of immune response.77FH13240754.1Attorney Docket No.: ABH-01225

[0272] In some embodiments, the cell comprising the GPC3-targeted CAR is a cytotoxic T cell or CD8+T cell. Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL- 10, adenosine and other molecules secreted by regulatory T cells, the CD8+cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0273] In some embodiments, the cell comprising the GPC3-targeted CAR is a memory T cell. Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon reexposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0274] In some embodiments, the cell comprising the GPC3-targeted CAR is a regulatory T cell. Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Treg cells have been described — naturally occurring Tregcells and adapti ve Tregcells.

[0275] In some embodiments, the cell comprising the GPC3-targeted CAR is a Natural killer T cell. Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CDld.

[0276] In some embodiments, the cell comprising the GPC3-targeted CAR is a y5 T cell. y5 T cells possess a distinct T-cell receptor (TCR) having one y chain and one 5 chain instead of a and chains.

[0277] In some embodiments, the cell comprising the GPC3-targeted CAR is an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, or a lymphokine activated killer (LAK) cell.

[0278] In some embodiments, the cell is a T cell, and the GPC3-targeted CAR is integrated at a locus within the genome of the T cell. Non-limiting examples of the loci 78FH13240754.1Attorney Docket No.: ABH-01225include a TRAC locus, a TRBC locus, a TRDC locus, and a TRGC locus. In some embodiments, the locus is a TRAC locus or a TRBC locus. Methods of targeting a CAR to a site within the genome of T cell are disclosed in WO2017180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113-117, both of which are incorporated by reference in their entireties.

[0279] In some embodiments, the T cells comprise a mixture of CD4+T cells and CD8+T cells. In some embodiments, the T cells are enriched for one or more subsets based on cell surface marker expression profile.

[0280] In some embodiments, the cell is a Natural killer cell. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells. Unlike cytotoxic CD8+T cells, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization and can also eradicate MHC-I-negati ve cells. In some embodiments, the cell is a genetically modified NK cell. In some embodiments, the cell is an edited NK cell. In some embodiments, the cell is a NK cell derived from a stem cell. In some embodiments, the cell is a NK cell derived from a pluripotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC)-derived NK cell.

[0281] The cells (e.g., T cells or NK cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells. In some embodiments, the cells comprising a GPC3-targeted CAR are obtained from the subject to be treated (i.e. are autologous). In some embodiments, the cells comprising a GPC3-targeted CAR are allogeneic to the subject to be treated.

[0282] The cells can be cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cell, erythrocyte, thrombocytes, and stem cells from which myeloid cells may be differentiated. In some embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0283] Epstein-Barr virus (EBV)-induced lymphoproliferative diseases (EBV-LPDs) and other EBV-associated cancers are a significant cause of morbidity and mortality for recipients of allogeneic hematopoietic cell transplantation (HCT) or solid organ transplants (SOT), particularly in those who have received certain T-cell reactive Abs to prevent or treat GVHD. Prophylaxis and treatment by the adoptive transfer of autologous or allogeneic EBV-specific 79FH13240754.1Attorney Docket No.: ABH-01225cytotoxic T cells and the subsequent long-term restoration of immunity against EBV-associated lymphoproliferation have provided positive outcomes in the management of these uniformly fatal complications of allogeneic tissue transfer. Therefore, in some embodiments, the disclosed immune effector cells that comprise one or more of the CAR polypeptides described herein are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, generation of EBV-specific cytotoxic T cells may involve isolating PBMCs from of an EBV-seropositive autologous or allogenic donor and enriching them for T cells by depletion of monocytes and NK cells. EBV-specific cytotoxic T cells may also be produced by contacting donor PBMCs or purified donor T cells with a “stimulator” cell that expresses one or more EBV antigen(s) and presents the EBV antigen(s) to unstimulated T cells, thereby causing stimulation and expansion of EBV-specific CTLs. EBV antigens include, for example, latent membrane protein (LMP) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A, and LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. Cytotoxic T cells that comprise T cell receptor(s) which recognize one or more EBV-specific antigens are deemed to have been “sensitized” to those EBV antigen(s) and are therefore termed “EBV-sensitized cytotoxic T cells” herein. Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may comprise one or more of the CAR polypeptides are described, for example, in Barker et al., Blood 116(23):5045-49 (2010); Doubrovinaet al., Blood 119(11):2644-56 (2012); Koehne et al., Blood 99(5): 1730-40 (2002); and Smith et al., Cancer Res. 72(5): 1116-25 (2012), which are incorporated by reference for these teachings. Similarly, cytotoxic T cells may be “sensitized” to other viral antigens, including cytomegalovirus (CMV), papillomavirus (e.g., HPV), adenovirus, polyomavirus (e.g., BKV, JCV, and Merkel cell virus), retrovirus (e.g., HTLV-I, also including lentivirus such as HIV), picomavirus (e.g., Hepatitis A virus), hepadnavirus (e.g., Hepatitis B virus), hepacivirus (e.g., Hepatitis C virus), deltavirus (e.g., Hepatitis D virus), hepevirus (e.g., Hepatitis E virus), and the like. In some embodiments, the T cells used for generating the CAR-T cells are polyfunctional T-cells, i.e., those T cells that are capable of inducing multiple immune effector functions, that provide a more effective immune response to a pathogen than do cells that produce, for example, only a single immune effector (e.g. a single biomarker such as a cytokine or CD107a). Less-polyfunctional, monofunctional, or even “exhausted” T cells may dominate immune responses during chronic infections, thus negatively impacting protection against virus-associated complications. In some embodiments, the CAR-T cells are polyfunctional. In 80FH13240754.1Attorney Docket No.: ABH-01225some embodiments, at least 50% of the T cells used for generating the CAR-T cells are CD4+T cells. In some embodiments, the T cells are less than 50% CD4+T cells. In some embodiments, the T cells are predominantly CD4+T cells. In some embodiments, at least 50% of the T cells used for generating the CAR-T cells are CD8+T cells. In some embodiments, the T cells are less than 50% CD8+T cells. In some embodiments, the T cells are predominantly CD8+T cells. In some embodiments, the T cells (e.g., the sensitized T cells and / or CAR-T cells described herein) are stored in a cell library or bank before they are administered to the subject.

[0284] In some embodiments, the cells can be transduced with the GPC3-targeted CAR such that the cells express the GPC3-targeted CAR.Anti-GPC3 Antibodies

[0285] Provided herein are binding agents or fragments thereof that bind to GPC3. In some embodiments, the binding agents or fragments thereof are anti-GPC3 antibodies and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, multispecific antibodies, bispecific antibodies, or hetero-conjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0286] In some embodiments, the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody and antigen-binding fragment thereof, binds to human GPC3.Monospecific Antibodies and Antigen-binding Fragments Thereof

[0287] In some embodiments, the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0288] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 139; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 140. SEQ ID NO: 139 and SEQ ID NO: 140 are disclosed in Table 1.

[0289] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising81FH13240754.1Attorney Docket No.: ABH-01225the amino acid sequence of SEQ ID NO: 360. SEQ ID NO: 359 and SEQ ID NO: 360 are disclosed in Table 2.

[0290] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 361. SEQ ID NO: 359 and SEQ ID NO: 361 are disclosed in Table 3.

[0291] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 359; and / or ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 362. SEQ ID NO: 359 and SEQ ID NO: 362 are disclosed in Table 4.

[0292] The VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 can be identified according to any known CDR numbering systems, which include but are not limited to, Kabat numbering system, Chothia numbering system, AbM numbering system. Contact numbering system, and IMGT® Information System. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are identified according to the Kabat numbering system.

[0293] In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133 or a conservative modification thereof. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136 or a conservative modification thereof. In some embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133. In some embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136.82FH13240754.1Attorney Docket No.: ABH-01225

[0294] In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 358, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 133; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the CDRs are identified according to the Kabat numbering system. SEQ ID NO: 358, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136 are disclosed in Tables 1-4.

[0295] In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 137; and / or a VL comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138.

[0296] In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 359; and / or a VL comprising an amino acid sequence that is at least about 80%, at least about 85%,83FH13240754.1Attorney Docket No.: ABH-01225at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 360. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 360. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 360.

[0297] In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 359; and / or a VL comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 361.

[0298] In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 359;84FH13240754.1Attorney Docket No.: ABH-01225and / or a VL comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the binding agent or fragment thereof that bind to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 362.

[0299] The antibody or antigen-bi tiding fragment thereof, e.g., the murine antibody or antigen-binding fragment thereof, designated as “GPC3-8” comprises the VH CDR1, VH CDR2, and VH CDR3 sequences disclosed in Table 1; and the VL CDR1, VL CDR2, and VL CDR3 sequences disclosed in Table 1. The GPC3-8 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 (disclosed in Table 1) and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138 (disclosed in Table 1).

[0300] The antibody or antigen-bi tiding fragment thereof, e.g., the humanized antibody or antigen-binding fragment thereof, designated as “GPC3-8-hl” comprises the VH CDR1, VH CDR2, and VH CDR3 sequences disclosed in Table 2; and the VL CDR1, VL CDR2, and VL CDR3 sequences disclosed in Table 2. The GPC3-8-hl antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 (disclosed in Table 2) and a VL comprising the amino acid sequence set forth in SEQ ID NO: 360 (disclosed in Table 2).

[0301] The antibody or antigen-bi tiding fragment thereof, e.g., the humanized antibody or antigen-binding fragment thereof, designated as “GPC3-8-h2” comprises the VH CDR1, VH CDR2, and VH CDR3 sequences disclosed in Table 3; and the VL CDR1, VL CDR2, and VL CDR3 sequences disclosed in Table 3. The GPC3-8-h2 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 (disclosed in Table 3) and a VL comprising the amino acid sequence set forth in SEQ ID NO: 361 (disclosed in Table 3).

[0302] The antibody or antigen-bi tiding fragment thereof, e.g., the humanized antibody or antigen-binding fragment thereof, designated as “GPC3-8-h3” comprises the VH CDR1, VH CDR2, and VH CDR3 sequences disclosed in Table 4; and the VL CDR1, VL CDR2, and VL 85FH13240754.1Attorney Docket No.: ABH-01225CDR3 sequences disclosed in Table 4. The GPC3-8-h3antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 359 (disclosed in Table 4) and a VL comprising the amino acid sequence set forth in SEQ ID NO: 362 (disclosed in Table 4).

[0303] In some embodiments, the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, comprises a fragment crystallizable region (Fc region). Fc region is the C-terminal region of an immunoglobulin heavy chain. The Fc region can be a functional Fc region, a native Fc region, a recombinant Fc region, or a variant Fc region. In some embodiments, the Fc region comprises one or more heavy chain constant domains (e.g., CH2, and CH3). In some embodiments, the variant Fc region comprises one or more modifications (e.g., substitutions) in the CH2 domain. In some embodiments, the variant Fc region comprises one or more modifications (e.g., substitutions) in the CH3 domain. In some embodiments, the heavy chain constant region is chosen from IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. In some embodiments, the heavy chain constant region is chosen from IgGl, IgG2, IgG3, and IgG4. In some embodiments, the heavy chain constant region is chosen from IgGl . In some embodiments, the heavy chain constant region is chosen from human IgGl.

[0304] In some embodiments, the Fc region is an IgGl Fc region. In some embodiments, the Fc region is a human IgGl Fc region. In some embodiments, the Fc region is a native human IgGl Fc region. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 357. SEQ ID NO: 357 is provided below.DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO : 357 ]

[0305] In some embodiments, the Fc region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 357.

[0306] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding 86FH13240754.1Attorney Docket No.: ABH-01225region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

[0307] A “native Fc region” encompasses a Fc region found in nature, and not manipulated, modified, and / or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Nonlimiting examples of native human Fc regions include a native human IgGl Fc region (non- A and A allotypes), a native human IgG2 Fc region, a native human IgG3 Fc region a native human IgG4 Fc region as well as naturally occurring variants of the foregoing.

[0308] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) preferably one or more amino acid substitution(s). In some embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native Fc region or to the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, from about 1 to about 5 amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region described herein can possess at least about 80%, at least about 90%, at least about 95% or at least about 99% sequence identity with a native Fc region and / or with an Fc region of a parent polypeptide. A variant Fc region may comprises a loss of effector function (e.g., silent Fc).

[0309] The constant region / framework region of the antibodies can be altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation, etc., the number of cysteine residues, effector cell function, effector cell function, complement function or introduction of a conjugation site).

[0310] In some embodiments, the binding agent or fragment thereof that binds to GPC3 is an anti-GPC antibody. In some embodiments, the anti-GPC3 antibody is a human antibody. In some embodiments, the anti-GPC3 antibody is a humanized antibody. In some embodiments, the anti-GPC3 antibody is a monoclonal antibody. In some embodiments, the anti-GPC3 antibody is a human monoclonal antibody. In some embodiments, the anti-GPC3 antibody is a humanized monoclonal antibody.

[0311] In some embodiments, the binding agent or fragment thereof that bind to GPC3 is an anti-GPC antigen-binding fragment. In some embodiments, the antigen-binding fragment87FH13240754.1Attorney Docket No.: ABH-01225is a Fab, Fab’, F(ab’)2, Fv, or single chain variable fragment (scFv). In some embodiments, an antigen-binding fragment is an scFv.

[0312] In some embodiments, the antigen-binding fragment is an scFv-Fc. A scFv-Fc fragment comprises an scFv that is connected to a Fc region.Multispecific Molecules

[0313] Also provided herein are multispecific molecules comprising a binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or an antigen-binding portion thereof disclosed herein. In some embodiments, the multispecific molecules are multispecific antibodies. In some embodiments, the multispecific molecules (e.g., multispecific antibodies) are bispecific molecules (e.g., bispecific antibodies).

[0314] In some embodiments, the multispecific molecule binds to at least two different binding sites or target molecules. In some embodiments, the multispecific molecule comprises at least a first binding specificity for a GPC3 integrin, and a second binding specificity for a second target epitope. The second target epitope can be a GPC3 epitope, or a non-GPC3 epitope, e.g., an epitope on a second target antigen different from GPC3. In some embodiments, the non-GPC3 epitope is the second target antigen is tumor antigen.

[0315] The binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, can be derivatized or linked to more than one other functional molecule to generate multispecific molecules. To create a nuiltispecific molecule, a binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or an antigen-binding fragment thereof, can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a multispecific molecule results.

[0316] The multispecific molecules can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SAT A), 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidom ethyl) cyclohaxane-l-carboxylate (sulfo-SMCC).88FH13240754.1Attorney Docket No.: ABH-01225

[0317] Other formats of bispecific antibodies can be constructed, such tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb), and fusion protein with human serum albumin (Ryutaro, et al., J Biol Chem 2011; 286: 1812-1818; Anja, et al., Blood 2000; 95(6): 2098-2103; Weiner, et al., J. Immunology 1994; 152(5): 2385-2392; Dafne, et al., J Biol Chem 2007; 282: 12650-12660), but are devoid of Fc effector functions with distinct pharmacokinetic profiles.

[0318] Methods for making multispecific molecules are known in the art, such as, by coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities. Exemplary structures of multispecific molecules are known in the art.

[0319] For example, bispecific molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. In some embodiments, BslgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in Kk-bodies), and use of heterodimeric Fc regions. Strategies are known in the art to avoid heavy chain pairing of homodimers in BslgG, including knobs-into-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity.

[0320] Another bispecific molecule format is IgG appended with an additional antigenbinding moiety. For example, monospecific IgG can be engineered to have bi-specificity by appending an additional antigen-binding unit onto the monospecific IgG, for example, at the N- or C- terminus of either the heavy or light chain.

[0321] Bispecific antibody fragments (BsAb) are a format of bispecific molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In some embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody.89FH13240754.1Attorney Docket No.: ABH-01225

[0322] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. For example, bispecific antibody fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. In some embodiments, chemical conjugation, for example, chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules.

[0323] Methods of production of multispecific molecules, including bispecific molecules, are known in the art. For example, multispecific molecules, including bispecific molecules, can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly or by expression of the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) molecules can be performed by various methods known in the art, including affinity chromatography.Cross-competing Antibodies

[0324] Additionally, provided herein are binding agents or fragments thereof that bind to GPC3, e.g., antibodies or antigen-binding fragments thereof, that cross-compete for binding to a GPC3 antigen (e.g., a human GPC3 antigen) with any of the binding agents or fragments thereof that bind to GPC3, e.g., anti-GPC3 antibodies or antigen-binding fragments thereof (e.g., GPC3-8, GPC3-8-hl, GPC3-8-h2, and GPC3-8-h3). For example, the cross-competing antibodies bind to the same epitope region, e.g., same epitope, adjacent epitope, or overlapping as any of the anti-GPC3 antibodies or antigen-binding fragments thereof (e.g., GPC3-8, GPC3-8-hl, GPC3-8-h2, and GPC3-8-h3).

[0325] The competition can be determined by an assay in which the test antibody under study prevents or inhibits the specific binding of the reference antibody to a common epitope or a common antigen (e.g., a GPC3 antigen). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to a5 integrin (e.g., human GPC3). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory 90FH13240754.1Attorney Docket No.: ABH-01225Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., (1990) Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., a5 integrin, such as human a5 integrin) bound to a solid surface or cells bearing either of an un -labelled test antigen binding protein (e.g., test a5 integrin antibody) or a labeled reference antigen binding protein (e.g., reference anti-a5 integrin antibody).Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and / or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur (e.g., similar epitope or overlapping epitope). Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common epitope or common antigen by at least about 20%, for example, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% or at least about 97%, at least about 98%, or at least about 99% or more.Immunoconjugates

[0326] Further provided are conjugates comprising a binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof. In some embodiments, the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, is conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as “immunoconjugates.” Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Non-limiting examples include taxol (such as ricin, diphtheria, gelonin), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and 91FH13240754.1Attorney Docket No.: ABH-01225puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, calecheamicin, aureastatin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.g., vincristine and vinblastine).

[0327] Other non-limiting examples of therapeutic cytotoxins that can be conjugated to the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. A non-limiting example of a calicheamicin antibody conjugate is commercially available (Mylotarg™; Wyeth-Ayerst).

[0328] Cytoxins can be conjugated to the binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

[0329] The binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to,90Y,131I,225Ac,213Bi,223Ra,177Lu, and227Th. Method for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the anti-GPC3 antibody or antigen-binding fragment thereof.92FH13240754.1Attorney Docket No.: ABH-01225

[0330] The conjugates can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or acti ve fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor (TNF) or interferon-y; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.Engineered and Modified Antibodies

[0331] The binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, can be prepared using an antibody or an antigen-binding fragment thereof comprising one or more of the VHand / or VLsequences disclosed herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the start! ng antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VHand / or VL), for example within one or more CDR regions and / or within one or more framework regions. Additionally, or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

[0332] One type of variable region engineering that can be performed is CDR grafting.

[0333] Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321 :522-525; Queen, C. et al. (1989) Proc. Nad. Acad. See. U.S.A. 86: 10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)93FH13240754.1Attorney Docket No.: ABH-01225

[0334] Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. The VHCDR1, CDR2, and CDR3 sequences, and the VLCDR1, CDR2, and CDR3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences.

[0335] Another type of variable region modification is to mutate amino acid residues within the VHand / or VLCDR1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays. In some embodiments, conservative modifications are introduced. The mutations may be amino acid substitutions, additions or deletions. For example, no more than one, two, three, four or five residues within a CDR region are altered.

[0336] Accordingly, the embodiments described herein provide binding agents or fragments thereof that bind to GPC3, e.g., anti-GPC3 antibodies or antigen-binding fragments thereof, comprising a VHcomprising: (a) a CDR1 sequence of the antibodies and antigen-binding fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VHCDR1 sequence of any one of the antibodies or antigen-binding fragments thereof disclosed herein; (b) a CDR2 sequence of any one of the antibodies or antigen-binding fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VHCDR2 of any one of the antibodies or antigen-binding fragments thereof disclosed herein; and (c) a CDR3 sequence of any one of the antibodies or antigen-binding fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VHCDR3 of any one of the antibodies or antigen-binding fragments thereof disclosed herein; and aVLcomprising (a) a CDR1 sequence of any one of the antibodies or antigen-binding94FH13240754.1Attorney Docket No.: ABH-01225fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VL CDR1 of any one of the antibodies or antigen-binding fragments thereof disclosed herein; (b) a CDR2 sequence of any one of the antibodies or antigen-binding fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VL CDR2 of any one of the antibodies or antigen-binding fragments thereof disclosed herein; and / or (c) a CDR3 sequence of any one of the antibodies or antigen-binding fragments thereof disclosed herein, or an amino acid sequence having at least one (e.g., no more than one, no more than two, no more than three, no more than four or no more than five) amino acid modification (e.g., substitution, deletion and / or addition) as compared to the VL CDR3 of any one of the antibodies or antigen-binding fragments thereof disclosed herein.

[0337] Engineered antibodies include those in which modifications are made to framework residues within VH and / or VL, e.g., to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

[0338] In addition or alternative to modifications made within the framework or CDR regions, the binding agents or fragments thereof that bind to GPC3, e.g., anti-GPC3 antibodies or antigen-binding fragments thereof, can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, an anti-GPC3 antibody or antigen-binding fragment thereof can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. The hinge region of CHI can be modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of 95FH13240754.1Attorney Docket No.: ABH-01225the light and heavy chains or to increase or decrease the stability of the antibody. The Fc hinge region of an antibody may be mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. The antibody can be modified to increase its biological half-life, e.g., the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG. Furthermore, the Fc region can be altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. The Fc region can be modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fey receptor. In some embodiments, an anti-GPC3 antibody comprises an afucosylated Fc region. Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) can result in a dramatic enhancement of ADCC through improved affinity for Fey receptor Ilia (FcyRIIIa).

[0339] Additionally or alternatively, the glycosylation of an anti-GPC3 antibody can be modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitution can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.

[0340] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery.

[0341] Another modification of the antibodies may be pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. In some embodiments, to pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or 96FH13240754.1Attorney Docket No.: ABH-01225antibody fragment. The pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Ci0) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody.Nucleic Acids and Vectors

[0342] In some embodiments, provided herein are nucleic acids encoding the binding molecules. In one aspect, nucleic acids encoding the GPC3-targeted CARs are provided, and cells comprising such nucleic acids. In some embodiments, the nucleic acid further comprises a promoter that is operably linked to the nucleic acid encoding a GPC3-targeted CAR.

[0343] In another aspect, provided herein are nucleic acids encoding the binding agents or fragments thereof that bind to GPC3, e.g., anti-GPC3 antibodies or antigen-binding fragments thereof, disclosed herein or a region thereof, and cells comprising such nucleic acids. In some embodiments, the nucleic acid comprises a first polynucleotide encoding an anti-GPC3 VH disclosed herein (e.g., one disclosed in Tables 1-4). In some embodiments, the first polynucleotide encodes an anti-GPC3 VH comprising the amino acid sequence set forth in SEQ ID NO: 137, or SEQ ID NO: 359. In some embodiments, the first polynucleotide encodes an anti-GPC3 VH comprising a CDR1, a CDR2, and a CDR3 disclosed in Tables 1-4.

[0344] In some embodiments, the nucleic acid comprises a second polynucleotide encoding an anti-GPC3 VL disclosed herein (e.g., one disclosed in Tables 1-4). In some embodiments, the second polynucleotide encodes an anti-GPC3 VL comprising the amino acid sequence set forth in SEQ ID NO: 138, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 362. In some embodiments, the second polynucleotide encodes an anti-GPC3 VL comprising a CDR1, a CDR2, and a CDR3 disclosed in Tables 1-4.

[0345] In some embodiments, the nucleic acid further comprises a promoter that is operably linked to the nucleic acid encoding a binding agent or fragment thereof that binds to GPC3, e.g., anti-GPC3 antibody or antigen-binding fragment thereof, or a region of an anti-GPC3 antibody or antigen-binding fragment thereof (e.g., VH or VL).97FH13240754.1Attorney Docket No.: ABH-01225

[0346] In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from the group consisting of an elongation factor (EF)-l promoter, a cytomegalovirus immediate-early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter, and Ubiquitin C promoter. In some embodiments, the endogenous promoter is selected from a TCR alpha promoter, a TCR beta promoter, and a beta 2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the group consisting of a NF AT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a4-lBB promoter, aPDl promoter, and aLAG3 promoter.

[0347] The presently disclosed embodiments also provide vectors comprising the nucleic acids. In some embodiments, the vector is an expression vector.

[0348] The nucleic acids can be delivered into cells by art-known methods or as described herein. Genetic modification of a cell can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In some embodiments, a retroviral vector (e.g., gammaretroviral vector or lentiviral vector) is employed for the introduction of the DNA construct into the cell. For example, a nucleic acid can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest.

[0349] Expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0350] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0351] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 98FH13240754.1Attorney Docket No.: ABH-01225Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vectors are lenti viral or retroviral vectors.

[0352] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.

[0353] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0354] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.

[0355] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene 99FH13240754.1Attorney Docket No.: ABH-01225or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.

[0356] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0357] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0358] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0359] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.100FH13240754.1Attorney Docket No.: ABH-01225

[0360] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0361] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a soludon containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0362] Any targeted genome editing methods can also be used to deliver a GPC3-targeted CAR to a cell. In some embodiments, a CRISPR system is used to deliver a GPC3-targeted CAR. In some embodiments, zine-finger nucleases are used to deliver a GPC3-targeted CAR. In some embodiments, a TALEN system is used to deliver a GPC3-targeted CAR.

[0363] Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR 101FH13240754.1Attorney Docket No.: ABH-01225RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). CRISPR / Cas9 often employs a plasmid to transfect the target cells. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in a cell. The repair template carrying CAR expression cassette need also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence. Multiple crRNA’ s and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.

[0364] A zine-finger nuclease (ZFN) is an artificial restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific DNA sequences which allows a zine-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs. The most common method to generate new zine-finger domain is to combine smaller zine-finger “modules” of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type Ils restriction endonuclease Fokl. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into genome. When the targeted sequence is cleaved by ZFNs, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, whereby the homologous DNA template is integrated into the genome.

[0365] Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of 102FH13240754.1Attorney Docket No.: ABH-01225these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), metallothionein promoters, or Ubiquitin C promoter), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0366] Methods for delivering the genome editing agents / systems can vary depending on the need. In some embodiments, the components of a selected genome editing method are delivered as DNA constructs in one or more plasmids. In some embodiments, the components are delivered via viral vectors. Common delivery methods include but is not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, replication-competent vectors cis and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic Nanoparticles, and cell-penetrating peptides).Methods of Detection

[0367] The disclosed embodiments provide methods for detecting GPC3 in a whole cell or tissue. In some embodiments, the method comprises: a) contacting a cell or tissue with an anti-GPC3 antibody or antigen-binding fragment thereof disclosed herein, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a detectable label; and b) determining the amount of the labeled anti-GPC3 antibody or antigen-binding fragment thereof bound to the cell or tissue. In some embodiments, the method is for detecting human GPC3.103FH13240754.1Attorney Docket No.: ABH-01225

[0368] In some embodiments, b) determining the amount of the labeled anti-GPC3 antibody or antigen-binding fragment thereof bound to the cell or tissue comprises measuring the amount of detectable label associated with the cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of GPC3 (e.g., human GPC3) in the cell or tissue.

[0369] The cell or tissue can be any cell or tissue, including any normal, healthy, abnormal, tumor, or cancer cells and tissues.

[0370] The disclosed embodiments also provide methods for detecting or diagnosing an GPC3-associated disease, disorder, or condition. A more definitive diagnosis of a GPC3-associated disease, disorder, or condition may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the a5 integrin-associated disease, disorder, or condition. In some embodiments, the method comprises: (a) measuring an expression level of GPC3 in a cell or a tissue sample of a subject using an anti-GPC3 antibody or antigen-binding fragment thereof disclosed herein; and (b) comparing the expression level of GPC3 measured in (a) with a control expression level of GPC3, wherein an increase in the expression level of GPC3 measured in (a) as compared to the control expression level of GPC3 is indicative of an GPC3-associated disease, disorder, or condition. In some embodiments, the control expression level of GPC3 is an expression level of GPC3 in a cell or a tissue sample of a subject not suffering from an GPC3-associated disease, disorder, or condition.Formulations and Administration

[0371] In yet another aspect, embodiments of the invention provide compositions comprising the cells comprising a GPC3-targeted CAR. In another aspect, the disclosed embodiments provide compositions comprising the anti-GPC3 antibodies or antigen-binding fragments thereof, the conjugates, or the multispecific molecules disclosed herein.

[0372] In some embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0373] The compositions can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous 104FH13240754.1Attorney Docket No.: ABH-01225compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0374] Compositions comprising the cells can be provided systemically or directly to a subject in need thereof. In some embodiments, the compositions are directly injected into an organ of interest. Alternatively, the compositions are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of compositions to increase production of cells in vitro or in vivo.

[0375] The quantity of cells comprising the GPC3-targeted CAR comprised in the compositions can vary for the subject being treated depending, for example, on the subject’s weight, size, age, etc along with the type and / or size and of their tumor and the stage of their disease. In some embodiments, the composition comprises between about IxlO4and about IxlO10, between about lxl04and about IxlO7, between about IxlO5and about IxlO7, between about IxlO5and about IxlO8, between about IxlO5and about 109, between about IxlO6and about IxlO8, between about IxlO6and about IxlO9, or between about IxlO6and about IxlO10of the cells comprising the GPC3-targeted CAR. In some embodiments, the composition comprises between about IxlO5and about 5xl08of the cells comprising the GPC3-targeted CAR. In some embodiments, the composition comprises between about IxlO5and about 1 xlO7of the cells comprising the GPC3-targeted CAR. In some embodiments, the composition comprises between about IxlO6and about 1 xlO8of the cells comprising the GPC3-targeted CAR. In some embodiments, the composition comprises between about IxlO6and about 5xl08of the cells comprising the GPC3-targeted CAR. More effective cells may be administered in even smaller numbers. Usually, at least about 1 x 105cells will be administered, eventually reaching about I x lO10or more. In some embodiments, the composition comprises at least about IxlO5, 5xl05, IxlO6, about 5xl06, about IxlO7, about 2.5xl07, about 5xl07, about IxlO8, about 1.5xl08, about 2xl08, or about 5xl08of the cells comprising the GPC3-targeted CAR. In some embodiments, the composition comprises about IxlO6of the cells comprising the GPC3-targeted CAR. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the 105FH13240754.1Attorney Docket No.: ABH-01225particular subject. Dosages can be readily ascertained by those skilled in the art from this specification and the knowledge in the art. The compositions can be administered multiple times at these dosages.

[0376] The CAR-modified cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2, IL-15, or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodiments formulated for intravenous administration. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0377] The administration of the compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions can be administered by any method known in the art including, but not limited to, intravenous administration, intradermal administration, subcutaneous administration, intranodal administration, intratumoral administration, intramedullary administration, intramuscular administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to the subject. In some embodiments, the composition is administered to a subject intravenously. The compositions can be injected directly into a tumor, lymph node, or site of disease.Methods of Treatment

[0378] The GPC3-targeted CAR-expressing cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions can be used for treating a GPC3-associated disease, disorder, or condition, including one or more symptoms of the disease, disorder, or condition. Thus, the disclosed embodiments provide methods of treating a GPC3-associated disease,106FH13240754.1Attorney Docket No.: ABH-01225disorder, or condition. In some embodiments, the method comprises administering to a subject in need thereof a binding agent or fragment thereof that binds GPC3 (e.g., anti-GPC3 antibody, antigen-binding fragment thereof, multispecific molecule), conjugate, or composition. In some embodiments, the method comprises administering to a subject in need thereof a composition comprising cells comprising a GPC3-targeted CAR. In some embodiments, the subject suffers from or is diagnosed with a GPC3-associated disease, disorder, or condition. In some embodiments, the subject is human.

[0379] Immune effector cells expressing the GPC3-targeted CARs can elicit an immune response against GPC3-expressing cells. The immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific to GPC3.

[0380] Adoptive transfer of immune effector cells expressing CARs is a promising anticancer therapeutic. Following the collection of a subject’s immune effector cells, the cells may be genetically engineered to express the GPC3-targeted CARs, then infused back into the subject. Moreover, cells (e.g., immune effector cells) obtained from a donor other than the subject (e.g., allogeneic to the subject) may be genetically engineered to express the GPC3-targeted CARs, then the CAR-expressing cells infused into the subject. In some embodiments, the cells comprising a GPC3-targeted CAR polypeptide are allogeneic EBV-specific cytotoxic T cells.

[0381] The disclosed embodiments provide various methods of using the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates, or compositions. The cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates, or compositions can be used in a therapy or medicament. For example, the disclosed embodiments provide methods for inducing and / or increasing an immune response in a subject in need thereof. The cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigenbinding fragments thereof), multispecific molecules, conjugates, or compositions can be used for treating a GPC3-associated disease or disorder in a subject. In some embodiments, the GPC3-associated disease or disorder in a subject is a tumor. Thus, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments 107FH13240754.1Attorney Docket No.: ABH-01225thereof), multispecific molecules, conjugates, or compositions can be used for reducing tumor burden in a subject, and / or prolonging the survival of a subject suffering from a tumor. The cells and compositions comprising thereof can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject. In some embodiments, each of the above-noted methods comprises administering the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates, or compositions to achieve the desired effect, e.g., palliation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0382] In some embodiments, the methods comprise administering to the subject a population of cells. In some embodiments, the population of cells comprises T cells. In some embodiments, at least a portion of the T cells expand in the subject after administration in response to a presence of GPC-3 expressing cells. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the T cells expand in the subject after administration in response to a presence of GPC-3 expressing cells. In some embodiments, at least a portion of the T cells expand in the subject after administration for a period of at least about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 2 months, about 3 months, or about 4 months. In some embodiments, at least a portion of the T cells expand in the subject after administration for a period of up to about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 2 months, about 3 months, or about 4 months. In some embodiments, at least a portion of the T cells expand in the subject after administration for a period of up to about 28 days. In some embodiments, at least a portion of the T cells expand in the subject for a period of up to about 49 days. In some embodiments, at least a portion of the T cells secrete IFNy when engaged with cells expressing GPC3. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 108FH13240754.1Attorney Docket No.: ABH-0122513%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the T cells secrete IFNy when engaged with cells expressing GPC3. In some embodiments, an amount of IFNy secreted by the at least a portion of the T cells peaks at about 1 to about 49 days, about 1 to about 42 days, about 1 to about 35 days, about 1 to about 28 days, about 1 to about 21 days, about 7 to about 49 days, about 7 to about 42 days, about 7 to about 35 days, about 7 to about 28 days, about 7 to about 21 days, about 14 to about 49 days, about 14 to about 42 days, about 14 to about 35 days, about 14 to about 28 days, or about 14 to about 21 days after administration. In some embodiments, an amount of IFNy secreted by the at least a portion of the T cells peaks at about 14 to about 28 days after administration. In some embodiments, the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein an amount of IFNy secreted by the at least a portion of the T cells decreases in response to an amount of tumor shrinkage. In some embodiments, the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein a peak amount of IFNy secreted by the at least a portion of the T cells correlates with a negative inflection point in tumor size. In some embodiments, the IFNy in whole blood samples are about 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1000 pg / mL, 2000 pg / mL, 3000 pg / mL, 4000 pg / mL, 5000 pg / mL, 6000 pg / mL, 7000 pg / mL, 8000 pg / mL, 9000 pg / mL, 10000 pg / mL, 15000 pg / mL, or 20000 pg / mL.

[0383] In one aspect, provides is a method of inhibiting growth of GPC3-expressing cells in a subject, the method comprising administering to the subject a cell disclosed herein, a binding agent or fragment thereof disclosed herein, a conjugate disclosed herein, a multispecific molecule disclosed herein, or a composition disclosed herein. In some embodiments, the GPC3-expressing cells are cancer cells, wherein the administered cell is a T cell and comprises a CAR, and the progression of growth of the GPC3-expressing cells has a negative inflection point at about 14 to about 21 days after administration of the cell. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 109FH13240754.1Attorney Docket No.: ABH-0122512%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition. In some embodiments, the cancerous tumor comprising the GPC3-expressing cells shrinks by about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition.

[0384] In some embodiments, the CARs or CAR-expressing cells inhibit growth of GPC3-expressing cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the CARs or CAR-expressing cells inhibit growth of GPC3-expressing cells by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%. In some embodiments, the GPC3-expressing cells are Huh7 cells. In some embodiments, the CAR prolongs proliferation of T cells transduced with the CAR in a subject by at least 7, at least 14, at least 21, or at least 28 days. In some embodiments, the CAR induces proliferation of T cells transduced with the CAR in a subject for at least 7, at least 14, at least 21, at least 28, at least 35, at least 42, or at least 49 days. In some embodiments, the CAR induces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold higher expansion of T cells transduced with the CAR compared to T-cells not transduced 110FH13240754.1Attorney Docket No.: ABH-01225with a CAR. In some embodiments, the CAR induces secretion of IFNy in a subject. In some embodiments, the CAR increases secretion of IFNy in a subject by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold.

[0385] In some embodiments, the disease or disorder is associated with GPC3 expression (i.e., it is a GPC3 -associated disorder). In some embodiments, the GPC3-associated disease or disorder overexpresses GPC3. Non-limiting examples of GPC3-associated disease or disorder include hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma. In some embodiments, the GPC3-associated disease or disorder is a tumor. In some embodiments, the GPC3-associated disease or disorder is cancer. In some embodiments, the GPC3-associated disease or disorder is hepatocellular carcinoma.

[0386] In some embodiments, the subject is a human subject. The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.

[0387] As a consequence of surface expression of a GPC3-targeted CAR, adoptively transferred cells are endowed with augmented and selective cytolytic activity at the tumor site. Furthermore, subsequent to their localization to tumor and their proliferation, the cells turn the tumor site into a highly conductive environment for a wide range of cells involved in the physiological anti-tumor response.

[0388] In some embodiments, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions are administered to a subject in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities. In some embodiments, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions can be used in combination with chemotherapy, radiation, immunosuppressive agents, immunoablative agents, cytoxin, and cytokines.

[0389] In some embodiments, the GPC3-associated disease or disorder is a neoplastic disease or disorder such as cancer. In some embodiments, the cancer can be any neoplasm or tumor for which radiotherapy is currently used as a treatment. Alternatively, the cancer can 111FH13240754.1Attorney Docket No.: ABH-01225be a neoplasm or tumor that is not sufficiently sensitive to radiotherapy using standard methods. A representative but non-limiting list of cancers that the disclosed cells, anti-GPC3 antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates and compositions can be used to treat include: hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma. Still other cancers are also considered for treatment by the disclosed cells, anti-GPC3 antibodies or antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions. In particular embodiments, resected and / or removed tissue from an individual patient’s tumor can be evaluated using methods described herein and / or known in the art (e.g., by genetic sequencing) to determine if it expresses GPC3 and at what levels. If the evaluated tissue does express GPC3 at sufficient levels, then one more of the disclosed cells, anti-GPC3 antibodies or antigen-binding fragments thereof, multispecific molecules, conjugates and compositions can be administered to the patient / subject to treat the cancer using dosages (e.g., from about IxlO4to about IxlO10cells) and methods described herein.

[0390] The cells, anti-GPC3 antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates and compositions can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.

[0391] According to one or more embodiments, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions can be used in combination with a checkpoint inhibitor (e.g., antibodies that block PD-1 as described below). The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC;CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1, an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but 112FH13240754.1Attorney Docket No.: ABH-01225are not limited to antibodies that block PD-1 (e.g., Nivolumab (B MS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (e.g., MDX-1105 (B MS-936559), MPDL3280A, MSB0010718C), PD-L2 (e.g., rHIgM12B7), CTLA-4 (e.g., Ipilimumab (MDX-010), Tremelimumab (e.g., CP-675,206)), IDO, B7-H3 (e.g., MGA271), B7-H4, TIM3, LAG-3 (e.g., BMS-986016). Techniques for combining CARs with checkpoint inhibitors in immune effector cells and use thereof for the treatment of various disorders are described, for example, in WO 2017 / 040945, which is incorporated by reference herein.

[0392] T cell receptor activation plus co-sti nmlation can help generate optimal “killer” CD8 T cell responses, which can be provided through ligation of tumor necrosis factor receptor family members, including 0X40 (CD134) and 4-1BB (CD137). 0X40 is of particular interest as treatment with an activating (agonist) anti-OX40 mAh augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors.

[0393] In some embodiments, methods of using the cells, binding agents or fragments thereof that bind GPC3 etc. to treat a neoplastic disease or condition such as cancer may also comprise administering to the subject a second therapeutic agent. The second therapeutic agent may be selected to have a synergistic and / or enhanced therapeutic effect when combined with cells, binding agents or fragments thereof that bind GPC3. In some embodiments, the second therapeutic agent is an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5 -fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0394] In some embodiments, the second therapeutic agent is an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.

[0395] In some embodiments, the second therapeutic agent is an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0396] In some embodiments, the second therapeutic agent is a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0397] In some embodiments, the second therapeutic agent is a growth factor inhibitor, such as an inhibitor of ErbBl (EGFR) (such as an EGFR antibody, e.g. zalutumumab,113FH13240754.1Attorney Docket No.: ABH-01225cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM 1 or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).

[0398] In some embodiments, the second therapeutic agent is a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0399] In some embodiments, the second therapeutic agent is a cytokine, growth factor, chemokine, or a combination thereof. Non-li miti ng examples of cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Non-limiting examples of chemokines include Glu-Leu-Arg (ELR)-negative chemokines such as IP- 10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0400] In some embodiments, the second therapeutic agent is a cell cycle control / apoptosis regulator (or “regulating agent”). A cell cycle control / apoptosis regulator may include molecules that target and modulate cell cycle control / apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055) . Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

[0401] In some embodiments, the second therapeutic agent is a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, an antiandrogene (such as flutaminde / eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy-progesterone / provera, megestrol acepate / megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as114FH13240754.1Attorney Docket No.: ABH-01225anastrazole / arimidex, aminoglutethimide / cytraden, exemestane) or a hormone inhibitor (such as octreotide / sandostatin).

[0402] In some embodiments, the second therapeutic agent is an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0403] Combined administration, as described above, may be simultaneous, separate, or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. Further in various embodiments, the second therapeutic agent can also be administered before or after administration of the cells, binding agents or fragments thereof that bind GPC3 etc. at a selected time interval(s) e.g., hours or days before or after the administration of the cells, binding agents etc.

[0404] In some embodiments, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions are administered in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a subject is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-ill.

[0405] In some embodiments, the cells, binding agents or fragments thereof that bind GPC3 (e.g., anti-GPC3 antibodies, antigen-binding fragments thereof), multispecific molecules, conjugates and compositions are administered in combination with surgery.

[0406] The GPC3-targeted CAR-T cells may be designed in several ways that enhance tumor cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and prolong their therapeutic half-life. TRUCK (T-cells Redirected for Universal Cytokine Killing) T cells for example, possess a CAR but are also engineered to release cytokines such as IL- 12 that promote tumor killing. Because these cells are designed to release a molecular payload upon activation of the CAR once localized to the tumor environment, these CAR-T cells are sometimes also referred to as ‘armored CARs’. Several cytokines as cancer therapies are being investigated both pre-clinically and clinically, and may also prove useful when similarly incorporated into a TRUCK form of CAR-T therapy. Among these include IL-2, IL-3. IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-a, IFN-y, TNF-a, TRAIL, FLT3 ligand, Lymphotactin, and TGF- (Dranoff 2004). “Self- 115FH13240754.1Attorney Docket No.: ABH-01225driving” or “homing” CAR-T cells are engineered to express a chemokine receptor in addition to their CAR. As certain chemokines can be upregulated in tumors, incorporation of a chemokine receptor aids in tumor trafficking to and infiltration by the adoptive T-cell, thereby enhancing both specificity and functionality of the CAR-T (Moon 2011). Universal CAR-T cells also possess a CAR, but are engineered such that they do not express endogenous TCR (T-cell receptor) or MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signaling repertoire of the adoptive T-cell therapy prevents graft-versus-host-disease and rejection, respectively. Armored CAR-T cells are additionally so named for their ability to evade tumor immunosuppression and tumor-induced CAR-T hypofunction. These particular CAR-Ts possess a CAR, and may be engineered to not express checkpoint inhibitors. Alternatively, these CAR-Ts can be co-administered with a monoclonal antibody (mAb) that blocks checkpoint signaling. Administration of an anti-PD-L1 antibody significantly restored the killing ability of CAR TILs (tumor infiltrating lymphocytes). While PD1-PDL1 and CTLA-4-CD80 / CD86 signaling pathways have been investigated, it is possible to target other immune checkpoint signaling molecules in the design of an armored CAR-T including LAG-3, Tim-3, IDO-1, 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin-ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase) . Armored CAR-Ts may also be engineered to express proteins or receptors that protect them against or make them resistant to the effects of tumor-secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with the double negative form of the TGF- receptor are resistant to the immunosuppression by lymphoma secreted TGF- . These transduced cells sh...

Claims

Attorney Docket No.: ABH-01225WHAT IS CLAIMED IS:

1. A chimeric antigen receptor (CAR), the CAR comprising an extracellular domain that binds to GPC3, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 358, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 133, and the VL comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 136, wherein:(a) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 360;(b) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 361; or(c) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 362.129FH13240754.1Attorney Docket No.: ABH-012252. The CAR of claim 1, wherein:the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360;the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361; orthe VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

3. The CAR of claim 1 , wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360.

4. The CAR of claim 1 , wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361.

5. The CAR of claim 1, wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

6. The CAR of any one of claims 1-5, wherein the extracellular domain comprises a Fv, a scFv, a Fab, or a F(ab)2.

7. The CAR of claim 6, wherein the extracellular domain comprises a scFv.

8. The CAR of any one of claims 1-7, wherein the extracellular domain comprises a linker between the VH and the VL.

9. The CAR of claim 8, wherein the linker comprises the amino acid sequence of SEQ ID NO: 1.

10. The CAR of any one of claims 1-9, wherein the VH is positioned N-terminal to the VL.

11. The CAR of any one of claims 1-9, wherein the VLIS positioned N-terminal to the VH.

12. The CAR of claim 1, wherein the extracellular domain comprises an scFv comprising the amino acid sequence of any one of SEQ ID NOs: 363-365.130FH13240754.1Attorney Docket No.: ABH-0122513. The CAR of any one of claims 1-12, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, aPD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide, or any combination thereof.

14. The CAR of claim 13, wherein the transmembrane domain comprises a CD28 polypeptide.

15. The CAR of claim 14, wherein the CD28 polypeptide comprises the amino acid sequence set forth in amino acids 154 to 179 of SEQ ID NO: 308.

16. The CAR of any one of claims 1-15, wherein the intracellular domain comprises a CD3^ polypeptide.

17. The CAR of claim 16, wherein the CD3^ polypeptide is a modified CD3^ polypeptide.

18. The CAR of claim 17, wherein the modified CD3^ polypeptide comprises a native IT AMI, an ITAM2 variant comprising two loss-of-function mutations, and an ITAM3 comprising two loss-of-function mutations.

19. The CAR of claim 18, wherein the native IT AMI comprises the amino acid sequence of SEQ ID NO: 313.

20. The CAR of claim 18, wherein the ITAM2 variant comprises the amino acid sequence of SEQ ID NO: 319.

21. The CAR of claim 18, wherein the ITAM3 variant comprises the amino acid sequence of SEQ ID NO: 323.

22. The CAR of any one of claims 17-21, wherein the modified CD3^ polypeptide comprises the amino acid sequence of SEQ ID NO: 325.

23. The CAR of any one of claims 1-22, wherein the intracellular domain comprises at least one co- stimulatory signaling region.131FH13240754.1Attorney Docket No.: ABH-0122524. The CAR of claim 23, wherein the at least one co-stimulatory signaling region comprises at least one intracellular signaling domain of a co-stimulatory molecule.

25. The CAR of claim 24, wherein the co-stimulatory molecule is CD28, 4- IBB, 0X40, ICOS, DAP-10, CD27, CD28, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD8, CD4, B2C, CD80, CD86, DAP10, DAP12, MyD88, BTNL3NKG2D or combinations thereof.

26. The CAR of claim 25, wherein the co-stimulatory molecule is CD28.

27. The CAR of any one of claims 23-26, wherein the at least one co-stimulatory signaling region comprises amino acids 180 to 220 of SEQ ID NO: 308.

28. The CAR of any one of claims 1-27, comprising the amino acid sequence of SEQ ID NO: 367.

29. The CAR of any one of claims 1-28, comprising a spacer between the extracellular domain that binds to GPC3 and the transmembrane domain.

30. The CAR of claim 29, wherein the spacer comprises the amino acid sequence RAA.

31. The CAR of any one of claims 1-30, comprising a signal peptide.

32. The CAR of claim 31, wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 280.

33. The CAR of any one of claims 1-32, comprising a self-cleaving P2A peptide.

34. The CAR of claim 33, wherein the self-cleaving P2A peptide comprises the amino acid sequence set forth in SEQ ID NO: 329.

35. The CAR of claim 33 or 34, wherein the self-cleaving P2A peptide is at the C-terminus of the intracellular domain.

36. The CAR of any one of claims 1-32, wherein the CAR comprises a TGF dominant negative receptor (DNR).132FH13240754.1Attorney Docket No.: ABH-0122537. The CAR of claim 36, wherein the TGF DNR comprises the amino acid sequence of SEQ ID NO: 366.

38. The CAR of claim 36 or 37, wherein the CAR comprises a self-cleaving P2A peptide.

39. The CAR of claim 38, wherein the self-cleaving P2A peptide comprises the amino acid sequence set forth in SEQ ID NO: 329.

40. The CAR of claim 38 or 39, wherein the self-cleaving P2A peptide is at the C-terminus of the intracellular domain and links the TGF DNR to the intracellular domain.

41. The CAR of claim 1, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 368-370.

42. The CAR of any one of claims 1-41, wherein the CAR is expressed from a vector.

43. The CAR of claim 42, wherein the vector is a viral vector.

44. The CAR of claim 43, wherein the viral vector is a retroviral vector or a lenti viral vector.

45. The CAR of any one of claims 1-44, wherein the CAR inhibits growth of GPC3-expressing cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

46. The CAR of claim 45, wherein the GPC3-expressing cells are Huh7 cells.

47. The CAR of any one of claims 1-46, wherein the CAR prolongs proliferation of T cells transduced with the CAR in a subject by at least 7, at least 14, at least 21, or at least 28 days.

48. The CAR of any one of claims 1-46, wherein the CAR induces proliferation of T cells transduced with the CAR in a subject for at least 7, at least 14, at least 21, at least 28, at least 35, at least 42, or at least 49 days.

49. The CAR of any one of claims 1-46, wherein the CAR induces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- 133FH13240754.1Attorney Docket No.: ABH-01225fold, at least 10-fold, or at least 20-fold higher expansion of T cells transduced with the CAR compared to T-cells not transduced with a CAR.

50. The CAR of any one of claims 1-46, wherein the CAR induces secretion of IFNy in a subject.

51. The CAR of any one of claims 1 -46, wherein the CAR increases secretion of IFNy in a subject by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold.

52. A nucleic acid encoding the CAR of any one of claims 1-51.

53. The nucleic acid of claim 52, comprising a promoter operably linked to the sequence encoding the CAR.

54. The nucleic acid of claim 53, wherein the promoter is endogenous or exogenous.

55. The nucleic acid of claim 54, wherein the exogenous promoter is an elongation factor (EF)-l promoter, a cytomegalovirus immediate-early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter or a Ubiquitin C promoter.

56. The nucleic acid of claim 54, wherein the endogenous promoter is a TCR alpha promoter, a TCR beta promoter or a beta 2-microglobulin promoter.

57. The nucleic acid of claim 53, wherein the promoter is an inducible promoter.

58. The nucleic acid of claim 57, wherein the inducible promoter is a NF AT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4- IBB promoter, a PD1 promoter or a LAG3 promoter.

59. A vector comprising the nucleic acid of any one of claims 52-58.

60. The vector of claim 59, wherein the vector is a viral vector.

61. The vector of claim 60, wherein the viral vector is a retroviral vector or a lenti viral vector.134FH13240754.1Attorney Docket No.: ABH-0122562. A cell comprising the CAR of any one of claims 1-51, the nucleic acid of any one of claims 52-58, or the vector of any one of claims 59-61.

63. The cell of claim 62, wherein the cell is transduced with the CAR, the nucleic acid, or the vector.

64. The cell of claim 62 or claim 63, wherein the CAR is constitutively expressed on the surface of the cell.

65. The cell of any one of claims 62-64, wherein the cell is an immunoresponsive cell or an immune effector cell.

66. The cell of any one of claims 62-65, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

67. The cell of any one of claims 62-66, wherein the cell is a T cell, a B cell, a Natural Killer (NK) cell, a macrophage, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a lymphokine activated killer (LAK) cell, a stem cell from which a lymphoid cell may be differentiated, a stem cell from which a myeloid cell may be differentiated or a combination thereof.

68. The cell of claim 67, wherein the cell is a T cell.

69. The cell of claim 67 or 68, wherein the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell, a mucosal associated invariant T cell, a af> T cell or a y5 T cell.

70. The cell of claim 67, wherein the cell is a NK cell.

71. The cell of claim 70, wherein the NK cell is derived from a stem cell.

72. The cell of claim 71, wherein the stem cell is a pluripotent stem cell.

73. The cell of claim 67, wherein the cell is a pluripotent stem cell.

74. The cell of claim 72 or 73, wherein the pluripotent stem cell is an embryoid stem cell or an induced pluripotent stem cell.135FH13240754.1Attorney Docket No.: ABH-0122575. A binding agent, or a fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 358, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 133, and the VL comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 136, wherein:(a) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 360;(b) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 361; or(c) the VH comprises an amino acid sequence of SEQ ID NO: 359, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362, an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 362.

76. The binding agent or fragment thereof of claim 75, wherein:the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360;136FH13240754.1Attorney Docket No.: ABH-01225the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361; orthe VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

77. The binding agent or fragment thereof of claim 75, wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 360.

78. The binding agent or fragment thereof of claim 75, wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 361.

79. The binding agent or fragment thereof of claim 75, wherein the VH comprises an amino acid sequence of SEQ ID NO: 359, and the VL comprises an amino acid sequence of SEQ ID NO: 362.

80. The binding agent or fragment thereof of any one of claims 75-79, wherein the binding agent or fragment thereof specifically binds GPC3.

81. The binding agent or fragment thereof of any one of claims 75-80, wherein the binding agent or fragment thereof is or comprises an antibody or antigen-binding fragment.

82. The binding agent or fragment thereof of claim 81, wherein the antibody or antigen-binding fragment is monoclonal.

83. The binding agent or fragment thereof of claim 81 or 82, wherein the antibody or antigen-binding fragment is a chimeric, a humanized, or a human antibody or antigen-binding fragment.

84. The binding agent or fragment thereof of any one of claims 81-83, wherein the binding agent or fragment thereof is an antigen-binding fragment.137FH13240754.1Attorney Docket No.: ABH-0122585. The binding agent or fragment thereof of claim 84, wherein the antigen-binding fragment comprises a Fv, a scFv, a Fab, or a F(ab)2.

86. The binding agent or fragment thereof of claim 85, wherein the antigen-binding fragment comprises a scFv.

87. The binding agent or fragment thereof of claim 86, comprising a linker between the VH and the VL.

88. The binding agent or fragment thereof of claim 87, wherein the linker comprises the amino acid sequence of SEQ ID NO: 1.

89. The binding agent or fragment thereof of any one of claims 86-88, wherein the VH is positioned N-terminal to the VL.

90. The binding agent or fragment thereof of any one of claims 86-88, wherein the VL is positioned N-terminal to the VH.

91. The binding agent or fragment thereof of claim 75, comprising the amino acid sequence of any one of SEQ ID NOs: 363-365.

92. The binding agent or fragment thereof of any one of claims 81-83, wherein the antibody or antigen-binding fragment comprises an IgA, IgG, IgM, IgE, or IgD constant domain.

93. The binding agent or fragment thereof of claim 92, wherein the antibody or antigenbinding fragment comprises an IgG constant domain.

94. The binding agent or fragment thereof of claim 93, wherein the antibody or antigenbinding fragment comprises an IgGl, IgG2, IgG3, or IgG4 constant domain.

95. The binding agent or fragment thereof of claim 94, wherein the antibody or antigen-binding fragment comprises an IgGl constant domain.138FH13240754.1Attorney Docket No.: ABH-0122596. The binding agent or fragment thereof of claim 95, wherein the antibody or antigenbinding fragment comprises an IgGl Fc region having an amino acid sequence of SEQ ID NO: 357.

97. The binding agent or fragment thereof of any one of claims 75-96, which binds to GPC3 with a dissociation constant (KD) of less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, or less than about 6 nM.

98. A nucleic acid that encodes the binding agent or fragment thereof of any one of claims 75-97.

99. The nucleic acid of claim 98, comprising a first polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 373, and a second polynucleotide comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 374-376.

100. The nucleic acid of claim 98, comprising a nucleic acid sequence of any one of SEQ ID NOs: 377-379.

101. A vector comprising the nucleic acid of any one of claims 98-100.

102. The vector of claim 101, which is an expression vector.

103. A host cell comprising the vector of claim 101 or 102.

104. A conjugate comprising the binding agent or fragment thereof of any one of claims 75-97.

105. The conjugate of claim 104, wherein the binding agent or fragment thereof is linked to a therapeutic agent, a detectable agent, or a diagnostic agent.

106. The conjugate of claim 105, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug.139FH13240754.1Attorney Docket No.: ABH-01225107. The conjugate of any one of claims 104-106, which is an immunoconjugate.

108. A multispecific molecule comprising the binding agent or fragment thereof of any one of claims 75-97, linked to a second functional moiety.

109. The multispecific molecule of claim 108, wherein the second functional moiety has a different binding specificity than the binding agent or fragment thereof.

110. A method of producing a binding agent or fragment thereof, the method comprising culturing the host cell of claim 103 under conditions which induce expression of the binding agent or fragment thereof in the host cell.

111. A method for detecting GPC3 in a whole cell or tissue, the method comprising: contacting a cell or tissue with the binding agent or fragment thereof of any one of claims 75-97, wherein the binding agent or fragment thereof comprises a detectable label; and determining the amount of the labeled binding agent or fragment thereof bound to the cell or tissue by measuring the amount of detectable label associated with the cell or tissue, wherein the amount of bound binding agent or fragment thereof indicates the amount of GPC3 in the cell or tissue.

112. A composition comprising the cell of any one of claims 62-74, the binding agent or fragment thereof of any one of claims 75-97, the conjugate of any one of claims 104-107, or the multispecific molecule of claim 108 or 109.

113. The composition of claim 112, comprising a pharmaceutically acceptable carrier.

114. The composition of claim 112 or claim 113, comprising between about 1 x 105and about 5 x 108cells.

115. A method of treating a subject having a disease or disorder associated with expression of GPC3, the method comprising administering to the subject the cell of any one of claims 62-74, the binding agent or fragment thereof of any one of claims 75-97, the conjugate of any one of claims 104-107, the multispecific molecule of claim 108 or 109, or the composition of any one of claims 112-114.140FH13240754.1Attorney Docket No.: ABH-01225116. The method of claim 115, wherein the disease or disorder is a neoplastic disease or disorder.

117. The method of claim 115 or claim 116, wherein the disease or disorder is cancer.

118. The method of any one of claims 115-117, wherein the disease or disorder is hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, or urothelial carcinoma.

119. The method of claim 118, wherein the disease or disorder is hepatocellular carcinoma.

120. The method of any one of claims 115-119, wherein the subject is a human subject.

121. The method of any one of claims 115-120, comprising administering to the subject a population of cells, wherein each cell is a cell according to any one of claims 62-74.

122. The method of claim 121, wherein the population of cells comprises T cells.

123. The method of claim 122, wherein at least a portion of the T cells expand in the subject after administration in response to a presence of GPC-3 expressing cells.

124. The method of claim 122 or 123, wherein at least a portion of the T cells expand in the subject after administration for a period of up to about 28 days.

125. The method of claim 124, wherein at least a portion of the T cells expand in the subject for a period of up to about 49 days.

126. The method of any one of claims 122-125 wherein at least a portion of the T cells secrete IFNy when engaged with cells expressing GPC3.

127. The method of claim 126, wherein an amount of IFNy secreted by the at least a portion of the T cells peaks at about 14 to about 28 days after admi ni strati on.141FH13240754.1Attorney Docket No.: ABH-01225128. The method of claim 126, wherein the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein an amount of IFNy secreted by the at least a portion of the T cells decreases in response to an amount of tumor shrinkage.

129. The method of claim 126, wherein the disease or condition comprises a cancerous tumor expressing GPC-3 and wherein a peak amount of IFNy secreted by the at least a portion of the T cells correlates with a negative inflection point in tumor size.

130. A method of inhibiting growth of GPC3-expressing cells in a subject, the method comprising administering to the subject the cell of any one of claims 62-74, the binding agent or fragment thereof of any one of claims 75-97, the conjugate of any one of claims 104-107, the multispecific molecule of claim 108 or 109, or the composition of any one of claims 112-114.

131. The method of claim 130, wherein the GPC3-expressing cells are cancer cells, wherein the administered cell is aT cell and comprises the CAR of any one of claims 1-51, and wherein the progression of growth of the GPC3-expressing cells has a negative inflection point at about 14 to about 21 days after administration of the cell.

132. The method of claim 130, wherein a cancerous tumor comprising the GPC3-expressing cells shrinks in size in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition.

133. The method of claim 132, wherein the tumor shrinks by at least 90% in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition.

134. The method of claim 133, wherein the tumor shrinks by at least 95% in response to the administration of the cell, binding agent, conjugate, multispecific molecule or composition.

135. A method for producing a cell comprising the CAR of any one of claims 1-51, the method comprising introducing into a cell a nucleic acid that encodes the CAR of any one of claims 1-51.142FH13240754.1Attorney Docket No.: ABH-01225136. The method of claim 135, wherein the method comprises a contacting the cell with a viral vector comprising the nucleic acid that encodes the CAR.

137. The cell of any one of claims 62-74, the binding agent or fragment thereof of any one of claims 75-97, the conjugate of any one of claims 104-107, the multispecific molecule of claim 108 or 109, or the composition of any one of claims 112-114 for use in therapy.

138. The cell of any one of claims 62-74, the binding agent or fragment thereof of any one of claims 75-97, the conjugate of any one of claims 104-107, the multispecific molecule of claim 108 or 109, or the composition of any one of claims 112-114 for use in a method for treating a subject for a disease or disorder associated with expression of GPC3.

139. The use of claim 138, wherein the disease or disorder is a tumor.

140. The use of claim 138, wherein the disease or disorder is cancer.

141. The use of any one of claims 138-140, wherein the disease or disorder is hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma or urothelial carcinoma.

142. The use of claim 141, wherein the disease or disorder is hepatocellular carcinoma.

143. The use of any one of claims 138-142, wherein the subject is a human subject.

144. A method of treating hepatocellular carcinoma comprising delivering radiofrequency energy to ablate tissue at a target tumor site and administering a GPC3-targeted cell therapy in a subject.

145. The method of claim 144, wherein the GPC3-targeted cell therapy is administered after the delivery of radiofrequency energy.

146. The method of claim 145, wherein the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the delivery of radiofrequency energy.143FH13240754.1Attorney Docket No.: ABH-01225147. The method of any one of claims 144-146, wherein the GPC3-targeted cell therapy is administered intratumorally.

148. The method of any one of claims 144-147, wherein the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3.

149. The method of claim 148, wherein the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg.

150. The method of any one of claims 144-149, wherein the delivery of radiofrequency energy releases tumor-associated antigens to prime an immune response in the subject.

151. The method of any one of claims 144-150, wherein the delivery of radiofrequency energy heats tissue at the target tumor site to a temperature between about 60°C and about 100°C.

152. The method of any one of claims 144-151, wherein the delivery of radiofrequency energy is performed percutaneously under ultrasound guidance.

153. The method of any one of claims 144-152, comprising administering one or more immune checkpoint inhibitors.

154. The method of any one of claims 144-153, comprising administering one or more cytokines to enhance immune activation in the subject.

155. The method of any one of claims 144-154, comprising administering one or more adjuvants to enhance immune response in the subject.

156. A method of reducing tumor recurrence in a liver cancer in a subject by sequentially administering radiofrequency ablation therapy and GPC3-targeted cell therapy.

157. The method of claim 156, wherein the GPC3-targeted cell therapy is administered after the radiofrequency ablation therapy.144FH13240754.1Attorney Docket No.: ABH-01225158. The method of claim 157, wherein the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation therapy.

159. The method of any one of claims 156-158, wherein the GPC3-targeted cell therapy is administered intratumorally.

160. The method of any one of claims 156-159, wherein the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3.

161. The method of claim 160, wherein the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg.

162. The method of any one of claims 156-161, wherein the radiofrequency ablation therapy releases tumor-associated antigens to prime immune response.

163. The method of any one of claims 156-162, wherein the radiofrequency ablation therapy heats tissue at a target tissue to a temperature between about 60°C and about 100°C.

164. The method of any one of claims 156-163, wherein the radiofrequency ablation therapy is performed percutaneously under ultrasound guidance.

165. The method of any one of claims 156-164, comprising administering one or more immune checkpoint inhibitors.

166. The method of any one of claims 156-165, comprising administering one or more cytokines to enhance immune activation.

167. The method of any one of claims 156-166, comprising administering one or more adjuvants to enhance immune response.

168. A method for increasing survival rate in a hepatocellular carcinoma patient, comprising administering combined radiofrequency ablation therapy and GPC 3 -targeted cell therapy.145FH13240754.1Attorney Docket No.: ABH-01225169. The method of claim 168, wherein the GPC3-targeted cell therapy is administered after the radiofrequency ablation therapy.

170. The method of claim 169, wherein the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation therapy.

171. The method of any one of claims 168-170, wherein the GPC3-targeted cell therapy is administered intratumorally.

172. The method of any one of claims 168-171, wherein the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3.

173. The method of claim 172, wherein the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg.

174. The method of any one of claims 168-173, wherein the radiofrequency ablation therapy releases tumor-associated antigens to prime immune response.

175. The method of any one of claims 168-174, wherein the radiofrequency ablation is performed at a temperature between about 60°C and about 100°C.

176. The method of any one of claims 168-175, wherein the radiofrequency ablation is performed percutaneously under ultrasound guidance.

177. The method of any one of claims 168-176, comprising administering one or more immune checkpoint inhibitors.

178. The method of any one of claims 168-177, comprising administering one or more cytokines to enhance immune activation.

179. The method of any one of claims 168-178, comprising administering one or more adjuvants to enhance immune response.146FH13240754.1Attorney Docket No.: ABH-01225180. A method for inducing systemic anti -tumor immunity in a hepatocellular carcinoma patient, comprising administering combined radiofrequency ablation therapy and GPC3-targeted cell therapy.

181. The method of claim 180, wherein the GPC3-targeted cell therapy is administered after the radiofrequency ablation.

182. The method of claim 181, wherein the GPC3-targeted cell therapy is administered within about 24 to about 72 hours after the radiofrequency ablation.

183. The method of any one of claims 180-182, wherein the GPC3-targeted cell therapy is administered intratumorally.

184. The method of any one of claims 180-183, wherein the GPC3-targeted cell therapy comprises CAR T-cells engineered to target GPC3.

185. The method of claim 184, wherein the CAR T-cells are administered at a dose of about 1 xlO6cells / kg to about 1 x 107cells / kg.

186. The method of any one of claims 180-185, wherein the radiofrequency ablation releases tumor-associated antigens to prime immune response.

187. The method of any one of claims 180-186, wherein the radiofrequency ablation therapy heats tissue at a target tissue to a temperature between about 60°C and about 100°C.

188. The method of any one of claims 180-187, wherein the radiofrequency ablation therapy is performed percutaneously under ultrasound guidance.

189. The method of any one of claims 180-188, comprising administering one or more immune checkpoint inhibitors.

190. The method of any one of claims 180-189, comprising administering one or more cytokines to enhance immune activation.147FH13240754.1Attorney Docket No.: ABH-01225191. The method of any one of claims 180-190, comprising administering one or more adjuvants to enhance immune response.

192. The method of any one of claims 144-191, wherein the GPC3-targeted cell therapy comprises a humanized GPC3-targeted molecule or a cell having a humanized GPC3-targeted molecule.

193. The method of claim 192, wherein the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).

194. A pharmaceutical composition comprising GPC3-targeted immune cells for use in combination with radiofrequency ablation therapy.

195. The pharmaceutical composition of claim 194, comprising immune modulators.

196. The pharmaceutical composition of claim 194 or 195, wherein the GPC3-targeted immune cells comprise a humanized GPC3-targeted molecule.

197. The pharmaceutical composition of claim 196, wherein the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).

198. A kit comprising radiofrequency ablation equipment and GPC3-targeted cell therapy formulations.

199. A kit comprising formulations for radiofrequency ablation and GPC3-targeted cell therapy.

200. A use of radiofrequency ablation and GPC3-targeted cell therapy for treating advanced-stage liver cancer.

201. A therapeutic regimen combining radiofrequency ablation therapy and GPC 3 -targeted cell therapy for treatment of liver cancer in a subject.148FH13240754.1Attorney Docket No.: ABH-01225202. The kit of claim 198 or 199, use of claim 200, or therapeutic regimen of claim 201, wherein the GPC3-targeted cell therapy comprises a humanized GPC3-targeted molecule or a cell having a humanized GPC 3 -targeted molecule.

203. The kit, use, or therapeutic regimen of claim 202, wherein the humanized GPC3-targeted molecule comprises a binding agent or a fragment thereof, an antigen recognizing receptor, or a chimeric antigen receptor (CAR).149FH13240754.1

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